18232, a novel dual specificity phosphatase and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 18232 nucleic acid molecules, which encode novel dual specificity phosphatase family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 18232 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 18232 gene has been introduced or disrupted. The invention still further provides isolated 18232 proteins, fusion proteins, antigenic peptides and anti-18232 antibodies. Diagnostic methods utilizing compositions of the invention are also provided. The invention also provides methods of modulating the differentiation and proliferation of hematopoietic cells (e.g., erythroid cells) utilizing the compositions of the invention. Accordingly, methods of treating, preventing and/or diagnosing erythroid-associated disorders such as anemias, leukemias, and erythrocytosis are disclosed.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applicationnumber 60/185,772 filed on Feb. 29, 2000, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The intracellular phosphorylation of proteins is critical for aplethora of regulatory and signalling mechanisms in eukaryotic cells.Phosphorylation events can govern a wide range of cellular processes,including cell proliferation, differentiation, transcription, andmorphology. Serine/threonine protein kinases, also called serine proteinkinases, are frequently utilized in signalling cascades as the activityof these enzymes can be finely regulated by stimuli. A common stimulusis phosphorylation of the serine protein kinase itself. Hence,signalling pathways, such as the MAP protein kinase cascade and theJAK/STAT pathway, can contain multiple proteins kinases which aresequentially activated. Ultimately, kinase cascades can result in thephosphorylation of cytoskeletal proteins, transcription factors, andbiosynthetic enzymes. Another class of kinases includes the receptortyrosine kinases. Activated receptor tyrosine kinases not onlyautophosphorylate, but phosphorylate other intracellular signallingmolecules, including those specifically bound to autophosphorylatedreceptors.

[0003] An essential component of the aforementioned signalling pathwaysis the ability of the cell to desensitize, recycle, and counteractphosphorylation signals. The cell primarily utilizes enzymes, termedphosphatases, which remove the phosphate on tyrosine, serine, andthreonine side chains. Dual specificity phosphatases hydrolyzephosphotyrosine, phosphothreonine, and phosphoserine residues (for areview, see, e.g., Fauman and Saper (1996) Trends in Biochem. 21:412).This class of proteins is exemplified by the VH1 or vaccinia virus lateH1 gene protein, whose catalytic activity is required for vaccinia virusreplication. A human homolog of VH1, VHR, has also been identified.VH1-like dual specificity phosphatase can also include the phosphatasesPAC-1 and CL100/MKP-1, hVH-2/MKP-2, hVH-3, MKP-3, MKP-X, MKP-4, hVH-5,and M3/6 proteins. The PAC-1 and CL100 proteins hydrolyzephosphothreonine and phosphotyrosine residues on phosphorylated MAP(mitogen activated protein) kinases. In order to modulate signallingevents, the activity and expression of dual specificity phosphatases canbe finely regulated. For example, the PAC-1 and CL100 phosphatase can beinduced by growth factors (Keyse, S (1995) Biochim. Biophys.Acta1265:152-160).

[0004] Thus, the function of dual specificity phosphatase proteins canbe critical for the regulation of cellular processes such asproliferation and differentiation. Given the important biological rolesand properties of such phosphatases, there exists a need for theidentification of novel genes encoding such proteins as well as for thediscovery of modulators of such molecules for use in regulating avariety of normal and/or pathological cellular processes.

SUMMARY OF THE INVENTION

[0005] The present invention is based, in part, on the discovery of anovel dual specificity phosphatase, referred to herein as “18232”nucleic acid and protein molecules. The nucleotide sequence of a cDNAencoding 18232 is shown in SEQ ID NO:1, and the amino acid sequence of a18232 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotidesequence of the coding region is depicted in SEQ ID NO:3.

[0006] Accordingly, in one aspect, the invention features a nucleic acidmolecule that encodes a 18232 protein or polypeptide, e.g., abiologically active portion of the 18232 protein. In a preferredembodiment, the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO:2. In other embodiments, theinvention provides isolated 18232 nucleic acid molecules having thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the sequenceof the DNA insert of the plasmid deposited with ATCC Accession Number______. In still other embodiments, the invention provides nucleic acidmolecules that are substantially identical (e.g., naturally occurringallelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQID NO:3, or the sequence of the DNA insert of the plasmid deposited withATCC Accession Number ______. In other embodiments, the inventionprovides a nucleic acid molecule that hybridizes under stringenthybridization conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number ______,wherein the nucleic acid encodes a full length 18232 protein or anactive fragment thereof.

[0007] In a related aspect, the invention further provides nucleic acidconstructs that include a 18232 nucleic acid molecule described herein.In certain embodiments, the nucleic acid molecules of the invention areoperatively linked to native or heterologous regulatory sequences. Alsoincluded are vectors and host cells containing the 18232 nucleic acidmolecules of the invention, e.g., vectors and host cells suitable forproducing 18232 nucleic acid molecules and polypeptides.

[0008] In another related aspect, the invention provides nucleic acidfragments suitable as primers or hybridization probes for the detectionof 18232-encoding nucleic acids.

[0009] In still another related aspect, isolated nucleic acid moleculesthat are antisense to a 18232 encoding nucleic acid molecule areprovided.

[0010] In another aspect, the invention features 18232 polypeptides andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of 18232-mediated or related disorders. In another embodiment,the invention provides 18232 polypeptides having a 18232 activity.Preferred polypeptides are 18232 proteins including at least one dualspecificity phosphatase catalytic domain, and, preferably, having a18232 activity, e.g., a 18232 activity as described herein.

[0011] In other embodiments, the invention provides 18232 polypeptides,e.g., a 18232 polypeptide having the amino acid sequence shown in SEQ IDNO:2; the amino acid sequence encoded by the cDNA insert of the plasmiddeposited with ATCC Accession Number ______; an amino acid sequence thatis substantially identical to the amino acid sequence shown in SEQ IDNO:2; or an amino acid sequence encoded by a nucleic acid moleculehaving a nucleotide sequence that hybridizes under stringenthybridization conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or 3, or the sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number ______,wherein the nucleic acid encodes a full length 18232 protein or anactive fragment thereof.

[0012] In a related aspect, the invention provides 18232 polypeptides orfragments operatively linked to non-18232 polypeptides to form fusionproteins.

[0013] In another aspect, the invention features antibodies andantigen-binding fragments thereof, that react with, or more preferablyspecifically bind 18232 polypeptides.

[0014] In another aspect, the invention provides methods of screeningfor compounds that modulate the expression or activity of the 18232polypeptides or nucleic acids.

[0015] In still another aspect, the invention provides a process formodulating 18232 polypeptide or nucleic acid expression or activity,e.g. using the screened compounds. In certain embodiments, the methodsinvolve treatment of conditions related to decreased activity orexpression of the 18232 polypeptides or nucleic acids, such asconditions involving aberrant cellular proliferation of a 18232expressing cell, e.g., a hematopoietic cell (e.g., an erythroid cell(e.g., an erythrocyte or an erythroblast), a CD34 positive cell, aglycophorin A-expressing cell, a megakaryocyte). The condition mayinvolve increased hematopoietic cell activity or proliferation as in thecase of leukemia, e.g., an erythroleukemia; or decreased hematopoieticcell differentiation as in the case of, e.g., an anemia.

[0016] In still another aspect, the invention features a method ofmodulating (e.g., enhancing or inhibiting) the proliferation, survival,and/or differentiation of a cell, e.g., a 18232-expressing cell, e.g., ahematopoietic cell (e.g., an erythroid cell, a bone marrow cell such asa CD34 positive cell, a megakaryocyte). The method includes contactingthe cell with an agent that modulates the activity or expression of a18232 polypeptide or nucleic acid, in an amount effective to modulatethe proliferation and/or differentiation of the cell.

[0017] In a preferred embodiment, the 18232 polypeptide has an aminoacid sequence identical to, or substantially identical to, SEQ ID NO:2.In other embodiments, the 18232 polypeptide is a fragment of at least15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2.

[0018] In a preferred embodiment, the 18232 nucleic acid has anucleotide sequence identical to, or substantially identical to, SEQ IDNO:1 or 3. In other embodiments, the 18232 nucleic acid is a fragment ofat least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or morecontiguous nucleotides of SEQ ID NO:1 or 3.

[0019] In a preferred embodiment, the agent modulates (e.g., increasesor decreases) protein phosphatase activity.

[0020] In a preferred embodiment, the agent modulates (e.g., increasesor decreases) expression of the 18232 nucleic acid by, e.g., modulatingtranscription, mRNA stability, etc.

[0021] In preferred embodiments, the agent is a peptide, aphosphopeptide, a small molecule, e.g., a member of a combinatoriallibrary, or an antibody, or any combination thereof. The antibody can beconjugated to a therapeutic moiety selected from the group consisting ofa cytotoxin, a cytotoxic agent and a radioactive metal ion.

[0022] In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or an 18232 nucleic acid, or anycombination thereof.

[0023] In a preferred embodiment, the agent is administered incombination with a cytotoxic agent.

[0024] In a preferred embodiment, the cell, e.g., the 18232-expressingcell, is a hematopoietic cell, e.g., a myeloid, lymphoid or erythroidcell, or a precursor cell thereof. Examples of such cells includemyelocytic cells (polymorphonuclear cells), erythrocytic cells,lymphocytes, monocytes, reticular cells, plasma cells andmegakaryocytes, as well as stem cells for the different lineages, andprecursors for the committed progenitor cells, for example, precursorsof blood cells (e.g., red blood cells, such as erythroblasts),macrophages (monoblasts), platelets (megakaryocytes), polymorphonuclearleucocytes (myeloblasts), and lymphocytes (lymphoblasts).

[0025] In a preferred embodiment, the cell, e.g., the 18232-expressingcell, is a bone marrow cell, e.g., a bone marrow CD34-expressing cell.Examples of CD34-expressing cells include immature hematopoieticprecursor cells, hematopoietic colony-forming cells in bone marrow,including unipotent (CFU-GM, BFU-E) and pluripotent progenitors(CFU-GEMM, CFU-Mix and CFU-blast); as well as stromal cell precursors,terminal deoxynucleotidyl transferase (TdT) expressing B- and T-lymphoidprecursors, early myeloid cells and early erythroid cells.

[0026] In a preferred embodiment, the cell, e.g., the 18232-expressingcell, is a bone marrow erythroid cell, e.g., an erythroid progenitor(e.g., a glycophorin A expressing cell) or a differentiated cell, e.g.,an erythrocyte or a megakaryocyte.

[0027] In one embodiment, the hematopoietic cell is a lymphoid cell,e.g., B cells, and their precursors, T cells (e.g., CD4⁺8⁺ T cells,CD4⁺8⁻ T cells (e.g., helper T cells), CD4⁻CD8⁺ T cells (e.g., cytotoxicT cells), CD4⁻8⁻ T cells, and natural killer T cells) and theirprecursors.

[0028] In a preferred embodiment, the cell, e.g., the 18232-expressingcell, is further contacted with a protein, e.g., a cytokine or ahormone. Exemplary proteins include, but are not limited to, G-CSF,GM-CSF, stem cell factor, interleukin-3 (IL-3), IL-4, Flt-3 ligand,thrombopoietin, and erythropoietin. Most preferably, the protein iserythropoietin. The protein contacting step can occur before, at thesame time, or after the agent is contacted. The protein contacting stepcan be effected in vitro or ex vivo. For example, the cell, e.g., the18232-expressing cell is obtained from a subject, e.g., a patient, andcontacted with the protein ex vivo. The treated cell can bere-introduced into the subject. Alternatively, the protein contactingstep can occur in vivo.

[0029] In a preferred embodiment, the agent and the 18232-polypeptide ornucleic acid are contacted in vitro or ex vivo.

[0030] In a preferred embodiment, the contacting step is effected invivo in a subject, e.g., as part of a therapeutic or prophylacticprotocol. Preferably, the subject is a human, e.g., a patient with anerythroid-associated disorder. For example, the subject can be a patientwith an anemia, e.g., hemolytic anemia, aberrant erythropoiesis,secondary anemia in non-hematolic disorders, anemia of chronic diseasesuch as chronic renal failure; endocrine deficiency disease; and/orerythrocytosis (e.g., polycythemia). Alternatively, the subject can be acancer patient, e.g., a patient with leukemic cancer, e.g., an erythroidleukemia, or a carcinoma, e.g., a renal carcinoma. In other embodiments,the subject is a non-human animal, e.g., an experimental animal.

[0031] The contacting step(s) can be repeated.

[0032] In a preferred embodiment, the agent decreases the proliferationand/or enhances the differentiation of the cell, e.g., the18232-expressing cell, e.g., the erythroid cell. Such agents can be usedto treat or prevent cancers, e.g., leukemic cancers such as erythroidleukemias, or carcinomas, e.g., renal or lung carcinomas.

[0033] In a preferred embodiment, the agent increases the number oferythroid cells, by e.g., increasing the proliferation, survival, and/orstimulating the differentiation, of erythroid progenitor cells. Suchagents can be used to treat or prevent anemias, e.g., hemolytic anemias,aberrant erythropoiesis, secondary anemias in non-hematolic disorders,anemias of chronic diseases such as chronic renal failure; endocrinedeficiency diseases; and/or erythrocytosis (e.g., polycythemias).

[0034] In a preferred embodiment, the agent increases the number oferythroid cells, by e.g., increasing the proliferation, survival, and/orstimulating the differentiation, of granulocytic and monocyticprogenitor cells, e.g., CFU-GM, CFU-G (colony forming unit—granulocyte),myeloblast, promyelocyte, myelocyte, a metamyelocyte, or a band cell.Such compounds can be used to treat or prevent neutropenia andgranulocytopenia, e.g., conditions caused by cytotoxic chemotherapy,AIDS, congenital and cyclic neutropenia, myelodysplastic syndromes, oraplastic anemia.

[0035] In another aspect, the invention features a method of modulatinghematopoiesis, e.g., erythropoiesis, comprising contacting ahematopoietic cell, e.g., a blood cell, such as an erythroid cell, witha agent that increases or decreases the activity or expression of a18232 polypeptide or nucleic acid, thereby modulating thedifferentiation of the hematopoietic cell, e.g., the blood cell.

[0036] In a preferred embodiment, the 18232 polypeptide has an aminoacid sequence identical to, or substantially identical to, SEQ ID NO:2.In other embodiments, the 18232 polypeptide is a fragment of at least15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2.

[0037] In a preferred embodiment, the 18232 nucleic acid has anucleotide sequence identical to, or substantially identical to, SEQ IDNO:1 or 3. In other embodiments, the 18232 nucleic acid is a fragment ofat least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or morecontiguous nucleotides of SEQ ID NO:1 or 3.

[0038] In a preferred embodiment, the agent modulates (e.g., increasesor decreases) protein phosphatase activity.

[0039] In preferred embodiments, the agent is a peptide, aphosphopeptide, a small molecule, e.g., a member of a combinatoriallibrary, or an antibody, or any combination thereof. The antibody can beconjugated to a therapeutic moiety selected from the group consisting ofa cytotoxin, a cytotoxic agent and a radioactive metal ion.

[0040] In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or an 18232 nucleic acid, or anycombination thereof.

[0041] In a preferred embodiment, the agent is administered incombination with a cytotoxic agent.

[0042] In a preferred embodiment, the hematopoietic cell is an erythroidcell, e.g., an erythroid progenitor or differentiated cell, e.g., anerythrocyte or a megakaryocyte.

[0043] In a preferred embodiment, the hematopoietic cell is a bonemarrow CD34-expressing cell.

[0044] In a preferred embodiment, the agent and the 18232-polypeptide ornucleic acid are contacted in vitro or ex vivo.

[0045] In a preferred embodiment, the contacting step is effected invivo in a subject, e.g., as part of a therapeutic or prophylacticprotocol. Preferably, the subject is a human, e.g., a patient with ahematopoietic disorder such as an erythroid-associated disorder. Forexample, the subject can be a patient with an anemia, e.g., adrug-induced anemia (e.g., a chemotherapy-induced anemia), hemolyticanemia, aberrant erythropoiesis, secondary anemia in non-hematolicdisorders, anemia of chronic disease such as chronic renal failure;endocrine deficiency disease; and/or erythrocytosis (e.g.,polycythemia). Preferably, the erythroid-associated disorder is adrug-induced anemia (e.g., a chemotherapy induced anemia).Alternatively, the subject can be a cancer patient, e.g., a patient withleukemic cancer, e.g., an erythroid leukemia. In other embodiments, thesubject is a non-human animal, e.g., an experimental animal.

[0046] In a preferred embodiment, the method further includes contactingof the erythroid cell with a protein, e.g., a hormone. The protein canbe a member of the following non-limiting group: G-CSF, GM-CSF, stemcell factor, interleukin-3 (IL-3), IL-4, Flt-3 ligand, thrombopoietin,and erythropoietin. More preferably, the protein is erythropoietin. Theprotein contacting step can occur before, at the same time, or after theagent is contacted. The protein contacting step can be effected in vitroor ex vivo. For example, the cell, e.g., the erythroid cell can beobtained from a subject, e.g., a patient, and contacted with the proteinex vivo. The treated cell can be re-introduced into the subject.Alternatively, the protein contacting step can occur in vivo.

[0047] The contacting step(s) can be repeated.

[0048] In a preferred embodiment, the agent increases the number ofhematopoietic cells, e.g., erythroid cells, by e.g., increasing theproliferation, survival, and/or stimulating the differentiation, ofhematopoietic (e.g., erythroid) progenitor cells, in the subject. Suchagents can be used to treat an anemia, e.g., a drug- (e.g.,chemotherapy-) induced anemia, hemolytic anemia, aberranterythropoiesis, secondary anemia in non-hematolic disorder, anemia ofchronic diseases such as chronic renal failure; endocrine deficiencydisease; and/or erythrocytosis (e.g., polycythemias).

[0049] In a preferred embodiment, the agent increases the number oferythroid cells, by e.g., increasing the proliferation, survival, and/orstimulating the differentiation, of granulocytic and monocyticprogenitor cells, e.g., CFU-GM, CFU-G (colony forming unit—granulocyte),myeloblast, promyelocyte, myelocyte, a metamyelocyte, or a band cell.Such compounds can be used to treat or prevent neutropenia andgranulocytopenia, e.g., conditions caused by cytotoxic chemotherapy,AIDS, congenital and cyclic neutropenia, myelodysplastic syndromes, oraplastic anemia.

[0050] In yet another aspect, the invention features a method oftreating or preventing a hematopoietic disorder, e.g., anerythroid-associated disorder, in a subject. The method includesadministering to the subject an effective amount of an agent thatmodulates the activity or expression of a 18232 polypeptide or nucleicacid such that the hematopoietic disorder is ameliorated or prevented.

[0051] In a preferred embodiment, the 18232 polypeptide has an aminoacid sequence identical to, or substantially identical to, SEQ ID NO:2.In other embodiments, the 18232 polypeptide is a fragment of at least15, 20, 50, 100, 150, or more contiguous amino acids of SEQ ID NO:2.

[0052] In a preferred embodiment, the 18232 nucleic acid has anucleotide sequence identical to, or substantially identical to, SEQ IDNO:1 or 3. In other embodiments, the 18232 nucleic acid is a fragment ofat least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or morecontiguous nucleotides of SEQ ID NO:1 or 3.

[0053] In a preferred embodiment, the agent modulates (e.g., increasesor decreases) protein phosphatase activity.

[0054] In a preferred embodiment, the agent modulates (e.g., increasesor decreases) expression of the 18232 nucleic acid by, e.g., modulatingtranscription, mRNA stability, etc.

[0055] In preferred embodiments, the agent is a peptide, aphosphopeptide, a small molecule, e.g., a member of a combinatoriallibrary, or an antibody, or any combination thereof. The antibody can beconjugated to a therapeutic moiety selected from the group consisting ofa cytotoxin, a cytotoxic agent and a radioactive metal ion.

[0056] In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or an 18232 nucleic acid, or anycombination thereof.

[0057] In a preferred embodiment, the agent is administered incombination with a cytotoxic agent.

[0058] In a preferred embodiment, the subject is a human, e.g., apatient with a hematopoietic disorder, e.g., an erythroid-associateddisorder. For example, the subject can be a patient with an anemia,e.g., a drug- (chemotherapy-) induced anemia, hemolytic anemia, aberranterythropoiesis, secondary anemia in non-hematolic disorders, anemia ofchronic disease such as chronic renal failure; endocrine deficiencydisease; and/or erythrocytosis (e.g., polycythemia). Preferably, theanemia is a drug- (chemotherapy-) induced anemia. Alternatively, thesubject can be a cancer patient, e.g., a patient with leukemic cancer,e.g., an erythroid leukemia, or a patient with a carcinoma, e.g., arenal carcinoma. In other embodiments, the subject is a non-humananimal, e.g., an experimental animal.

[0059] In a preferred embodiment, the agent decreases the proliferationand/or enhances the differentiation of a cell, e.g., a 18232-expressingcell, e.g., a hematopoietic cell (e.g., an erythroid cell), in thesubject. Such agents can be used to treat or prevent cancers, e.g.,leukemic cancers such as erythroid leukemias, or carcinomas, e.g., renalcarcinomas.

[0060] In a preferred embodiment, the agent increases the number ofhematopoietic cells, e.g., blood cells (e.g., erythroid cells), by e.g.,increasing the proliferation, and/or stimulating the differentiation, oferythroid progenitor cells, in the subject. Such agents can be used totreat an anemia, e.g., a drug- (chemotherapy-) induced anemia, ahemolytic anemia, aberrant erythropoiesis, a secondary anemia innon-hematolic disorder, anemia of chronic diseases such as chronic renalfailure; endocrine deficiency disease; and/or erythrocytosis (e.g.,polycythemias).

[0061] In a preferred embodiment, the agent increases the number oferythroid cells, by e.g., increasing the proliferation, survival, and/orstimulating the differentiation, of granulocytic and monocyticprogenitor cells, e.g., CFU-GM, CFU-G (colony forming unit—granulocyte),myeloblast, promyelocyte, myelocyte, a metamyelocyte, or a band cell.Such compounds can be used to treat or prevent neutropenia andgranulocytopenia, e.g., conditions caused by cytotoxic chemotherapy,AIDS, congenital and cyclic neutropenia, myelodysplastic syndromes, oraplastic anemia.

[0062] In a preferred embodiment, the disorder is a hematopoieticdisorder, e.g. an erythroid-associated disorder. Examples oferythroid-associated disorder include drug- (chemotherapy-) inducedanemia, hemolytic anemia, aberrant erythropoiesis, secondary anemia innon-hematolic disorders, anemia of chronic disease such as chronic renalfailure; endocrine deficiency disease; and/or erythrocytosis (e.g.,polycythemia). Preferably, the erythroid associated disorder is a drug-(chemotherapy-) induced anemia.

[0063] In a preferred embodiment, the disorder is a cancer, e.g., aleukemic cancer, e.g., an erythroid leukemia, or a carcinoma, e.g., arenal carcinoma.

[0064] In a preferred embodiment, the method further includesadministering an effective amount of a protein, e.g., a cytokine or ahormone, to the subject. Exemplary proteins include, but are not limitedto, G-CSF, GM-CSF, stem cell factor, interleukin-3 (IL-3), IL-4, Flt-3ligand, thrombopoietin, and erythropoietin. Preferably, the protein iserythropoietin. The protein can be administered before, at the same timeor after, administration of the agent.

[0065] The administration of the agent and/or protein can be repeated.

[0066] In still another aspect, the invention features a method forevaluating the efficacy of a treatment of a disorder, in a subject. Themethod includes treating a subject with a protocol under evaluation;assessing the expression of a 18232 nucleic acid or 18232 polypeptide,such that a change in the level of 18232 nucleic acid or 18232polypeptide after treatment, relative to the level before treatment, isindicative of the efficacy of the treatment of the disorder.

[0067] In a preferred embodiment, the disorder is a hematopoieticdisorder, e.g., an erythroid-associated disorder. Examples oferythroid-associated disorders include an anemia, e.g., a drug- (e.g.,chemotherapy-) induced anemia, a hemolytic anemia, aberranterythropoiesis, secondary anemia in non-hematolic disorder, anemias ofchronic disease such as chronic renal failure; endocrine deficiencydiseases; and/or erythrocytosis (e.g., polycythemia).

[0068] In a preferred embodiment, the disorder is a cancer, e.g.,leukemic cancer, e.g., an erythroid leukemia, or a carcinoma, e.g., arenal carcinoma.

[0069] In a preferred embodiment, the subject is a human.

[0070] In a preferred embodiment, the subject is an experimental animal,e.g., an animal model for a hematopoietic- (e.g., an erythroid-)associated disorder.

[0071] In a preferred embodiment, the method can further includetreating the subject with a protein, e.g., a cytokine or a hormone.Exemplary proteins include, but are not limited to, G-CSF, GM-CSF, stemcell factor, interleukin-3 (IL-3), IL-4, Flt-3 ligand, thrombopoietin,and erythropoietin. Preferably, the protein is erythropoietin.

[0072] The invention also features a method of diagnosing a disorder,e.g., hematopoietic disorder (e.g., an erythroid-associated disorder),in a subject. The method includes evaluating the expression or activityof a 18232 nucleic acid or a 18232 polypeptide, such that, a differencein the level of 18232 nucleic acid or 18232 polypeptide relative to anormal subject or a cohort of normal subjects is indicative of thedisorder.

[0073] In a preferred embodiment, the subject is a human.

[0074] In a preferred embodiment, the evaluating step occurs in vitro orex vivo. For example, a sample, e.g., a blood sample, is obtained fromthe subject.

[0075] In a preferred embodiment, the evaluating step occurs in vivo.For example, by administering to the subject a detectably labeled agentthat interacts with the 18232 nucleic acid or polypeptide, such that asignal is generated relative to the level of activity or expression ofthe 18232 nucleic acid or polypeptide.

[0076] In a preferred embodiment, the disorder is a hematopoieticdisorder, e.g., a hematopoietic disorder as described herein.

[0077] In a preferred embodiment, the disorder is anerythroid-associated disorder, e.g., an erythroid-associated disorder asdescribed herein.

[0078] The invention also provides assays for determining the activityof or the presence or absence of 18232 polypeptides or nucleic acidmolecules in a biological sample, including for disease diagnosis.

[0079] In further aspect, the invention provides assays for determiningthe presence or absence of a genetic alteration in a 18232 polypeptideor nucleic acid molecule, including for disease diagnosis.

[0080] In yet another aspect, the invention features a method foridentifying an agent, e.g., a compound, which modulates the activity ofa 18232 polypeptide, e.g., a 18232 polypeptide as described herein, orthe expression of a 18232 nucleic acid, e.g., a 18232 nucleic acid asdescribed herein, including contacting the 18232 polypeptide or nucleicacid with a test agent (e.g., a test compound); and determining theeffect of the test compound on the activity of the polypeptide ornucleic acid to thereby identify a compound which modulates the activityof the polypeptide or nucleic acid.

[0081] In a preferred embodiment, the activity of the 18232 polypeptideis a protein phosphatase activity.

[0082] In a preferred embodiment, the activity of the 18232 polypeptideis hematopoiesis, e.g., erythropoiesis.

[0083] In a preferred embodiment, the activity of the 18232 polypeptideis proliferation, differentiation, and/or survival of a cell, e.g., a18232-expressing cell, e.g., a hematopoietic cell (e.g., a bone marrowcell such as a CD34 positive cell, an erythroid cell, a megakaryocyte).

[0084] In preferred embodiments, the agent is a peptide, aphosphopeptide, a small molecule, e.g., a member of a combinatoriallibrary, or an antibody, or any combination thereof.

[0085] In additional preferred embodiments, the agent is an antisense, aribozyme, or a triple helix molecule, or an 18232 nucleic acid, or anycombination thereof.

[0086] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0087]FIG. 1 depicts a cDNA sequence (SEQ ID NO:1) and predicted aminoacid sequence (SEQ ID NO:2) of human 18232. The methionine-initiatedopen reading frame of human 18232 (without the 5′ and 3′ untranslatedregions) starts at nucleotide 329 and ends at nucleotide 859 of SEQ IDNO:1 (shown also as coding sequence (SEQ ID NO:3)).

[0088]FIG. 2 depicts a hydropathy plot of human 18232. Relativehydrophobic residues are shown above the dashed horizontal line, andrelative hydrophilic residues are below the dashed horizontal line. Thecysteine residues (cys) are indicated by short vertical lines just belowthe hydropathy trace. The numbers corresponding to the amino acidsequence of human 18232 are indicated.

[0089]FIGS. 3A and 3B depict alignments of dual specificity phosphatasecatalytic domains (DSPc and dsp_(—)5, respectively) and of human 18232amino acid sequence with a consensus amino acid sequence derived from ahidden Markov model using PFAM (FIG. 3A) and SMART (FIG. 3B). The uppersequence is the consensus amino acid sequence (SEQ ID NOs:4 and 5,respectively), while the lower amino acid sequence corresponds to aminoacids 18 to 156 of SEQ ID NO:2.

[0090]FIG. 4 is a bar graph depicting relative 18232 mRNA expression asdetermined by TaqMan assays on mRNA derived from the human lung, colon,bone marrow cells (e.g., bone marrow glycophorin A (GPA)-expressingcells), peripheral blood cells, cord blood, and fetal liver. The highest18232 mRNA expression, i.e., greater than 450 relative units, wasobserved in skeletal muscle, cord blood (CD34+ cells), fetal liver(CD34+ cells), and bone marrow with low GPA levels and CD71 positive (BMGAPlo CD71+).

[0091]FIG. 5 is a bar graph depicting relative 18232 mRNA expression asdetermined by TaqMan assays on mRNA derived from human hematologicalsamples, e.g., bone marrow (BM), erythroid cells (ery), megakaryocytes(meg), neutrophils, and a negative reference sample (NTC). In somesamples, mRNA expression was detected at the indicated times in culture(e.g., 24 hrs., 48 hrs., days in culture). High levels of 18232 mRNAexpression, i.e., greater than 200 relative units, were observed in onesample of CD15+, Cd11B− bone marrow; in erythroid cells, especially day14 erythroid cells, glycophorin A or CD36+ erythroid cells; and in day12 and day 14 megakaryocytes.

[0092]FIG. 6 is a bar graph depicting relative 18232 mRNA expression asdetermined by TaqMan assays on mRNA derived from human tissue samples,both normal, and tumor. The samples are derived from epithelial cells,colon, kidney, liver, fetal liver, lung, spleen, tonsil, lymph node,thymus, endothelial cells, skeletal muscle, fibroblast, and skin. Thehighest relative expression among these samples was found in epithelialcells, kidney, fetal liver, lung, and skeletal muscle.

[0093]FIG. 7 is a bar graph depicting relative 18232 mRNA expression asdetermined by TaqMan assays on mRNA derived from adipose tissue,osteoblasts, aortic smooth muscle cells, and human umbilical veinendothelial cells (HUVEC).

[0094]FIG. 8 is a bar graph depicted relative 18232 mRNA expression asdetermined by TaqMan assays on mRNA derived from hematopoietic cells,including CD8+ cells, CD3+ cells, CD 14+ cells, peripheral blood cells,bone marrow cells, cord blood cells, and cells from cell culture lines,e.g., HepG2B, MA101, HL60, K562, Molt4, and Hep3B cells. High 18232expression was observed in glycophorin A positive bone marrow cells.

DETAILED DESCRIPTION

[0095] The human 18232 sequence (FIG. 1; SEQ ID NO:1), which isapproximately 1390 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 531nucleotides (nucleotides 329 to 859 of SEQ ID NO:1; SEQ ID NO:3). Thecoding sequence encodes a 176 amino acid protein (SEQ ID NO:2).

[0096] Human 18232 contains the following regions or other structuralfeatures:

[0097] a dual specificity phosphatase, catalytic domain (PF00782)located from about amino acid residue 18 to 156 of SEQ ID NO:2;

[0098] a “C—X₅—R” motif, located at about amino acid residues 103 to 109of SEQ ID NO:2, including an active site cysteine at about amino acid103 of SEQ ID NO:2, and an active site arginine at about amino acid 109of SEQ ID NO:2;

[0099] a VH1-like dual specificity phosphatase loop located at aboutamino acid residues 70 to 74 of SEQ ID NO:2, including a conservedgeneral acid, aspartic acid at about residue 74 of SEQ ID NO:2;

[0100] one predicted N-glycosylation site (PS00001) at amino acids 50 to53;

[0101] two predicted Protein Kinase C sites (PS00005) at about aminoacids 30 to 32 and 135 to 137 of SEQ ID NO:2;

[0102] three predicted N-myristylation sites (PS00008) from about aminoacid 29 to 34, 44 to 49, and 123 to 128 of SEQ ID NO:2; and

[0103] one amidation site (PS00009) at amino acids 6 to 9 of SEQ IDNO:2.

[0104] For general information regarding PFAM identifiers, PS prefix andPF prefix domain identification numbers, refer to Sonnhammer et al.(1997) Protein 28:405-420 and http://www.psc.edu/general/software/packages/pfam/pfam.html.

[0105] A plasmid containing the nucleotide sequence encoding human 18232was deposited with American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, on ______ and assignedAccession Number ______. This deposit will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. This deposit wasmade merely as a convenience for those of skill in the art and is not anadmission that a deposit is required under 35 U.S.C. §112.

[0106] The 18232 protein contains a significant number of structuralcharacteristics in common with members of the dual specificityphosphatase family. The term “family” when referring to the protein andnucleic acid molecules of the invention means two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin, or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

[0107] Dual specificity phosphatase proteins are characterized by acommon fold. Examples of members of the dual specificity phosphatasefamily include MAP kinase phosphatase-1 (MKP-1), which dephosphorylatesMAP kinase on both threonine and tyrosine residues and a human, vacciniaH1-related phosphatase (VHR), which also removes the phosphate fromphosphothreonine and phosphotyrosine residues. Dual specificityphosphatases are exemplified by the VH1 or vaccinia virus late H1 geneprotein, which hydrolyzes both phosphotyrosine, phosphothreonine, andphosphoserine. VH1 catalytic activity is required for viral replication.A human homolog of VH1, VHR, has been identified. The three dimensionalstructure of this family is based on models from x-ray crystallographicdata of protein tyrosine phosphatases, and human VHR. The VHR structureincludes a core domain consisting of a five-stranded mixed β-sheet andsix α-helices. This structure closely superimposes on the structure ofphosphotyrosine protein phosphatases. However, dual specificityphosphatases lack the KNRY motif, and the N-terminal structures oftyrosine protein phosphatases which endow these enzymes with a deepactive site specific for aryl phosphates. Thus, dual specificityphosphatases have a shallower active site relative to tyrosine proteinphosphatases and can accommodate phosphoserine and phosphothreoninesubstrates. Even so dual specificity phosphatases can have a greaterthan 50-fold faster rate of phosphatase activity for phosphotyrosinesubstrates than phosphothreonine or phosphoserine substrates.

[0108] Similar to the broader class of phosphatases, dual specificityphosphatases have a highly conserved active site including threecatalytic residues, a cysteine, an arginine, and an aspartic acid. Theactive site cysteine and arginine are found in the “C—X₅—R” motif of thetyrosine phosphatase signature (Prosite PS00383). This motif forms abinding pocket for three of the phosphate oxyanions. The cysteine actsas a nucleophile to accept the PO₃ group. The reaction transientlygenerates a phospho-cysteine intermediate before the phosphate istransferred to water. The active site arginine stabilizes thetransition-state by hydrogen bonding to phosphate oxygens. In additionthe histidine preceding the active site cysteine and the serine orthreonine following the active site arginine are responsible forlowering the pK_(a) of the cysteine to stabilize a negative charge onthe cysteine. The active site aspartic acid accelerates the reaction bydonating a protein to generate an uncharged hydroxyl (for a review, seeFauman and Saper (1996) Trends in Biochem. 21:412). A C—X₅—R motif isfound at about amino acids 150 to 156 of SEQ ID NO:2.

[0109] The 18232 proteins of the present invention show significanthomology to members of the dual specificity phosphatase family, and inparticular, vaccinia H1-related phosphatases (VHRs). Dual specificityphosphatases are known to play critical roles in growth factorsignaling. For example, VHR-like phosphatases are known todephosphorylate growth factor receptors and thereby eliminate theirsignaling. MAP-kinase phosphatases terminate MAP-kinase activity, thusleading to inhibition of growth factor-mediated mitogenic signaling.Thus, dual specificity phosphatases play a key role in inhibitingproliferation and stimulating the differentiation of cells. As the 18232proteins show homology to dual specificity phosphatases, these proteinsare likely to be involved in modulating (e.g., inhibiting) theproliferation and (e.g., stimulating) the differentiation of the cellsin which they are expressed, e.g., hematopoietic cells such as bloodcells, e.g., erythroid cells. Accordingly, the 18232 molecules of theinvention may be useful for developing novel diagnostic and therapeuticagents for 18232-mediated or related disorders, as described below.

[0110] A 18232 polypeptide of the invention can include a “dualspecificity phosphatase catalytic domain” or regions homologous with a“dual specificity phosphatase catalytic domain”. As used herein, theterm “dual specificity phosphatase catalytic domain” refers to an aminoacid sequence having about 50 to 200, preferably about 75 to 175, morepreferably about 100 to 150, and even more preferably about 130 to 145amino acid residues, and having a bit score for the alignment of thesequence to the dual specificity phosphatase domain (HMM) of the Pfamdatabase of at least 50, preferably 100, and more preferably 120, or 140or more. The dual specificity phosphatase catalytic domain (HMM) hasbeen assigned the PFAM Accession PF00782(http//genome.wustl.edu/Pfam/.html). An alignment of the dualspecificity phosphatase catalytic domain (amino acids 18-156 of SEQ IDNO:2) of human 18232 with a consensus amino acid sequence (SEQ ID NO:4)derived from a hidden Markov model in the Pfam database is depicted inFIG. 3A, and a similar alignment with a consensus amino acid sequence(SEQ ID NO:5) derived from the SMART database(http://smart.embl-heidelberg.de/) HMM model is depicted in FIG. 3B.

[0111] A dual specificity phosphatase domain preferable includes theconserved active site cysteine and arginine in a C—X₅—R motif found atabout amino acids 103 to 109 of SEQ ID NO:2. Preferably, the dualspecificity phosphatase domain includes a conserved general acid, e.g.,an aspartic acid which participates in catalysis. For example, a 18232protein has an aspartic acid located at about residue 74 of SEQ ID NO:2,located in a VH1-like dual specificity phosphatase loop located at aboutamino acid residues 70 to 74 of SEQ ID NO:2. Typically, dual specificityphosphatases are able to dephosphorylate tyrosine residues andserine/threonine residues.

[0112] In a preferred embodiment, a 18232 polypeptide or protein has a“dual specificity phosphatase catalytic domain” or a region thatincludes at least about 50 to 200, preferably about 75 to 175, morepreferably about 100 to 150, and even more preferably about 120 to 140amino acid residues and has at least about 70%, 80%, 90%, 95%, 99%, or100% homology with a “dual specificity phosphatase catalytic domain,”e.g., the dual specificity phosphatase catalytic domain of human 18232(e.g., residues 18 to 156 of SEQ ID NO:2).

[0113] To identify the presence of a “dual specificity phosphatasecatalytic domain” in a 18232 protein sequence and to make thedetermination that a polypeptide or protein of interest has a particularprofile, the amino acid sequence of the protein can be searched againsta database of HMMs (e.g., the Pfam database, release 2.1) using thedefault parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search).For example, the hmmsf program, which is available as part of the HMMERpackage of search programs, is a family specific default program forMILPAT0063 and a score of 15 is the default threshold score fordetermining a hit. Alternatively, the threshold score for determining ahit can be lowered (e.g., to 8 bits). A description of the Pfam databasecan be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and adetailed description of HMMs can be found, for example, in Gribskov etal.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl.Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol.235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, thecontents of which are incorporated herein by reference. A search wasperformed against the HMM database resulting in the identification of a“dual specificity phosphatase catalytic domain” in the amino acidsequence of human 18232 at about residues 18-156 of SEQ ID NO:2 (seeFIGS. 3A-3B).

[0114] A 18232 molecule can further include: at least one, preferablytwo protein kinase C phosphorylation sites; at least one, two, orpreferably three N-myristylation sites; preferably at least oneN-glycosylation site; and preferably at least one amidation site.

[0115] As used herein, “18232 activity”, “biological activity of 18232”or “functional activity of 18232”, refers to an activity exerted by a18232 protein, polypeptide or nucleic acid molecule on e.g., a18232-responsive cell or on a 18232 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 18232activity is a direct activity, such as an association with a 18232target molecule. A “target molecule” or “binding partner” is a moleculewith which a 18232 protein binds or interacts with in nature, e.g., aprotein containing one or more phosphotyrosine, phosphoserine, orphosphothreonine residues. A 18232 activity also can be an indirectactivity, e.g., a cellular signaling activity mediated by interaction ofthe 18232 protein with a 18232 receptor (e.g., a cytokine receptor).

[0116] Based on the above-described sequence similarities, the 18232molecules of the present invention are predicted to have similarbiological activities as dual specificity phosphatase family members.Dual specificity phosphatases are known to play critical roles in theregulation of numerous cellular processes, including hematopoiesis.These phosphatases have been shown to associate with activatedreceptors, e.g., cytokine receptors, and to modulate (e.g., inhibit)growth factor signaling by dephosphorylating receptors, e.g., cytokinereceptors, thereby negatively regulating mitogenic signaling. Proteinphosphatases have also been shown to regulate transcriptional activity,e.g., cytokine transcriptional activity, stimulated by protein kinasessuch as JAK/STAT and MAP kinase pathways. Therefore, proteinphosphatases are likely to be involved in regulating the signalingmachinery responsible for cell differentiation of, e.g., hematopoietic(e.g., blood) cells, and/or modulation, e.g., suppression, of apoptosis.

[0117] Based on the sequence similarity and tissue distributiondescribed below, the 18232 molecules of the invention are predicted tohave one or more of the following activities: (1) catalyze the removalof a phosphate group attached to a tyrosine residue in a protein target,e.g., a growth factor (e.g., cytokine) receptor; (2) catalyze theremoval of a phosphate group attached to a serine or threonine residuein a protein e.g., a growth factor (e.g., cytokine) receptor; (3)modulate growth factor (e.g., cytokine) activity; (4) modulate anintracellular signaling pathway, e.g., a MAP kinase, JAK/STAT, ERKkinase pathway; (5) modulate (e.g., stimulate) cell differentiation,e.g., differentiation of hematopoietic cells (e.g., differentiation ofblood cells (e.g., erythroid progenitor cells, such as CD34+ erythroidprogenitors)); (6) modulate hematopoiesis, e.g., erythropoiesis; (7)modulate cell proliferation, e.g., proliferation of hematopoietic cells(e.g., erythroid progenitor cells); (8) inactivate cell surface growthfactor receptors, e.g., cytokine receptors; (9) modulate apoptosis, of acell, e.g., increase apoptosis of a cancer cell, e.g., a leukemic cell,(e.g., an erythroleukemia cell); or suppress apoptosis of a blood orerythroid cell; or (10) modulate transcriptional activity, e.g.,cytokine transcriptional activity.

[0118] The molecules of the invention can be used to develop novelagents or compounds to treat and/or diagnose disorders involvingaberrant activities of the cells in which 18232 nucleic acids andpolypeptides are expressed. 18232 mRNA is found primarily inhematopoietic progenitor CD34 cells (FIGS. 4-7). Its expression isfurther enhanced in the erythroid lineage and increases as bonemarrow/blood cell differentiation proceeds. This pattern of expressionsuggests a role for 18232 in the regulation of cytokine signaling duringthe development of cells of the erythroid lineage.

[0119] More specifically, significant expression of 18232 mRNA is foundin the bone marrow, cord blood, fetal liver, and in particular, inCD34-expressing populations of cells from those tissues, such asmobilized peripheral blood CD34+ cells, normal adult bone marrow CD34+cells, cord blood CD34+ cells, normal adult bone marrow CD34+ cells, andfetal liver CD34+ cells; as well as erythroid progenitor cells, e.g.,bone marrow glycophorin A positive cells, erythropoietin treatederythroid burst forming units (BFUs), erythrocytes, in vitro generatederythroblasts, and megakaryocytes (FIGS. 4-7). 18232 mRNA is alsoexpressed in epithelial cells, kidney, skeletal muscle, fetal and adultheart, spinal cord, brain, lung, and cell lines K562, HepG2, Hep3B, HL60and Molt4. Thus, diagnostic and therapeutic methods using the moleculesof the invention (or agents that modulate the activity or expression ofthe 18232 molecules) to treat/diagnose disorders involving thecells/tissues expressing 18232 molecules are contemplated by the presentinvention.

[0120] As used herein, a “CD34-positive cell” or a “CD34-expressingcell” refers to a cell that expresses detectable levels of the CD34antigen, preferably human CD34 antigen. The sequence for human CD34 isprovided in SwissProt Accession Number P28906. The CD34 antigen istypically present on immature hematopoietic precursor cells andhematopoietic colony-forming cells in the bone marrow, includingunipotent (CFU-GM, BFU-E) and pluripotent progenitors (CFU-GEMM, CFU-Mixand CFU-blast). The CD34 is also expressed on stromal cell precursors.Terminal deoxynucleotidyl transferase (TdT)-positive B- and T-lymphoidprecursors in normal bone also are CD34+. The CD34 antigen is typicallypresent on early myeloid cells that express the CD33 antigen, but lackthe CD14 and CD15 antigens and on early erythroid cells that express theCD71 antigen and dimly express the CD45 antigen. The CD34 antigen isalso found on capillary endothelial cells and approximately 1% of humanthymocytes. Normal peripheral blood lymphocytes, monocytes, granulocytesand platelets do not express the CD34 antigen. CD34 antigen density ishighest on early hematopoietic progenitor cells and decreases as thecells mature. The antigen is undetectably on fully differentiatedhematopoietic cells. Approximately 60% of acute B-lymphoid leukemia'sand acute myeloid leukemia express the CD34 antigen. The antigen is notexpressed on chronic lymphoid leukemia (B or T lineage) or lymphomas.

[0121] As the 18232 polypeptides of the invention may modulate18232-mediated activities, they may be useful as of for developing noveldiagnostic and therapeutic agents for 18232-mediated or relateddisorders, e.g., blood cell- (e.g., erythroid-) associated disorders andother hematopoietic disorders.

[0122] As used herein, the term “erythroid associated disorders” includedisorders involving aberrant (increased or deficient) erythroblastproliferation, e.g., an crythroleukemia, and aberrant (increased ordeficient) erythroblast differentiation, e.g., an anemia.Erythrocyte-associated disorders include anemias such as, for example,drug- (chemotherapy-) induced anemias, hemolytic anemias due tohereditary cell membrane abnormalities, such as hereditaryspherocytosis, hereditary elliptocytosis, and hereditarypyropoikilocytosis; hemolytic anemias due to acquired cell membranedefects, such as paroxysmal nocturnal hemoglobinuria and spur cellanemia; hemolytic anemias caused by antibody reactions, for example tothe RBC antigens, or antigens of the ABO system, Lewis system, Iisystem, Rh system, Kidd system, Duffy system, and Kell system;methemoglobinemia; a failure of erythropoiesis, for example, as a resultof aplastic anemia, pure red cell aplasia, myelodysplastic syndromes,sideroblastic anemias, and congenital dyserythropoietic anemia;secondary anemia in non-hematolic disorders, for example, as a result ofchemotherapy, alcoholism, or liver disease; anemia of chronic disease,such as chronic renal failure; and endocrine deficiency diseases.

[0123] Agents that modulate 18232 polypeptide or nucleic acid activityor expression can be used to treat anemias, in particular, drug-inducedanemias or anemias associated with cancer chemotherapy, chronic renalfailure, malignancies, adult and juvenile rheumatoid arthritis,disorders of hemoglobin synthesis, prematurity, and zidovudine treatmentof HIV infection. A subject receiving the treatment can be additionallytreated with a second agent, e.g., erythropoietin, to further amelioratethe condition.

[0124] As used herein, the term “erythropoietin” or “EPO” refers to aglycoprotein produced in the kidney, which is the principal hormoneresponsible for stimulating red blood cell production (erythrogenesis).EPO stimulates the division and differentiation of committed erythroidprogenitors in the bone marrow. Normal plasma erythropoietin levelsrange from 0.01 to 0.03 Units/mL, and can increase up to 100 to1,000-fold during hypoxia or anemia. Graber and Krantz, Ann. Rev. Med.29:51 (1978); Eschbach and Adamson, Kidney Intl. 28:1 (1985).Recombinant human erythropoietin (rHuEpo or epoietin alpha) iscommercially available as EPOGEN.RTM. (epoietin alpha, recombinant humanerythropoietin) (Amgen Inc., Thousand Oaks, Calif.) and as PROCRIT.RTM.(epoietin alpha, recombinant human erythropoietin) (Ortho Biotech Inc.,Raritan, N.J.).

[0125] Another example of an erythroid-associated disorder iserythrocytosis. Erythrocytosis, a disorder of red blood celloverproduction caused by excessive and/or ectopic erythropoietinproduction, can be caused by cancers, e.g., a renal cell cancer, ahepatocarcinoma, and a central nervous system cancer. Diseasesassociated with erythrocytosis include polycythemias, e.g., polycythemiavera, secondary polycythemia, and relative polycythemia.

[0126] Aberrant expression or activity of the 18232 molecules may beinvolved in neoplastic disorders. Accordingly, treatment, prevention anddiagnosis of cancer or neoplastic disorders related to hematopoieticcells and, in particular, cells of the erythroid lineage are alsoincluded in the present invention. Such neoplastic disorders areexemplified by erythroid leukemias, or leukemias of erythroid precursorcells, e.g., poorly differentiated acute leukemias such aserythroblastic leukemia and acute megakaryoblastic leukemia. Additionalexemplary myeloid disorders include, but are not limited to, acutepromyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97). In particular, AML can include theuncontrolled proliferation of CD34+ cells such as AML subtypes M1 andM2, myeloblastic leukemias with and without maturation, and AML subtypeM6, erythroleukemia (Di Guglielmo's disease). Additional neoplasticdisorders include a myelodysplastic syndrome or preleukemic disorder,e.g., oligoblastic leukemia, smoldering leukemia. Additional cancers ofthe erythroid lineage include erythroblastosis, and other relevantdiseases of the bone marrow.

[0127] The term “leukemia” or “leukemic cancer” is intended to have itsclinical meaning, namely, a neoplastic disease in which white corpusclematuration is arrested at a primitive stage of cell development. Thedisease is characterized by an increased number of leukemic blast cellsin the bone marrow, and by varying degrees of failure to produce normalhematopoietic cells. The condition may be either acute or chronic.Leukemias are further typically categorized as being either lymphocytici.e., being characterized by cells which have properties in common withnormal lymphocytes, or myelocytic (or myelogenous), i.e., characterizedby cells having some characteristics of normal granulocytic cells. Acutelymphocytic leukemia (“ALL”) arises in lymphoid tissue, and ordinarilyfirst manifests its presence in bone marrow. Acute myelocytic leukemia(“AML”) arises from bone marrow hematopoietic stem cells or theirprogeny. The term acute myelocytic leukemia subsumes several subtypes ofleukemia: myeloblastic leukemia, promyelocytic leukemia, andmyelomonocytic leukemia. In addition, leukemias with erythroid ormegakaryocytic properties are considered myelogenous leukemias as well.

[0128] The molecules of the invention may also modulate the activity ofneoplastic, non-hematopoietic tissues in which they are expressed, e.g.,kidney, lung, liver, skeletal muscle. For example, increase expressionof 18232 molecules is detected on lung tumors compared to the normallung. Accordingly, the 18232 molecules can act as novel diagnostictargets and therapeutic agents for controlling one or more of cellularproliferative and/or differentiative disorders. Examples of suchcellular proliferative and/or differentiative disorders include cancer,e.g., carcinoma, sarcoma, or metastatic disorders. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of lung, prostate, colon, breast, and liver origin.

[0129] As used herein, the terms “cancer”, “hyperproliferative”, and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

[0130] The terms “cancer” or “neoplasms” include malignancies of thevarious organ systems, such as those affecting lung, breast, thyroid,lymphoid, gastrointestinal, and the genito-urinary tract. The terms“cancer” or “neoplasms” also includes adenocarcinomas that includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,cancer of the small intestine, and cancer of the esophagus.

[0131] The term “carcinoma” is art recognized and refers to malignanciesof epithelial or endocrine tissues including respiratory systemcarcinomas, gastrointestinal system carcinomas, genitourinary systemcarcinomas, testicular carcinomas, breast carcinomas, prostaticcarcinomas, endocrine system carcinomas, and melanomas. Exemplarycarcinomas include those forming from tissue of the cervix, lung,prostate, breast, head and neck, colon, and ovary. The term alsoincludes carcinosarcomas, e.g., malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures.

[0132] The term “sarcoma” is art recognized and refers to malignanttumors of mesenchymal derivation.

[0133] The 18232 protein, fragments thereof, and derivatives and othervariants of the sequence in SEQ ID NO:2 thereof are collectivelyreferred to as “polypeptides or proteins of the invention” or “18232polypeptides or proteins”. Nucleic acid molecules encoding suchpolypeptides or proteins are collectively referred to as “nucleic acidsof the invention” or “18232 nucleic acids.” 18232 molecules refer to18232 nucleic acids, polypeptides, and antibodies.

[0134] As used herein, the term “nucleic acid molecule” includes DNAmolecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., anmRNA) and analogs of the DNA or RNA generated, e.g., by the use ofnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

[0135] The term “isolated or purified nucleic acid molecule” includesnucleic acid molecules that are separated from other nucleic acidmolecules that are present in the natural source of the nucleic acid.For example, with respect to genomic DNA, the term “isolated” includesnucleic acid molecules that are separated from the chromosome with whichthe genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences that naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

[0136] As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. A preferred example of stringenthybridization conditions includes hybridization in 6X sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2X SSC, 0.1% SDS at 50° C. Another example of stringenthybridization conditions are hybridization in 6X sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in 0.2XSSC, 0.1% SDS at 55° C. A further example of stringent hybridizationconditions are hybridization in 6X sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2X SSC, 0.1% SDS at60° C. Preferably, stringent hybridization conditions are hybridizationin 6X sodium chloride/sodium citrate (SSC) at about 45° C., followed byone or more washes in 0.2X SSC, 0.1% SDS at 65° C. Particularlypreferred stringency conditions (and the conditions that should be usedif the practitioner is uncertain about what conditions should be appliedto determine if a sequence is within the invention) are 0.5M SodiumPhosphate, 7% SDS at 65° C., followed by one or more washes at 0.2X SSC,1% SDS at 65° C. Preferably, an isolated nucleic acid molecule of theinvention that hybridizes under stringent conditions to the sequence ofSEQ ID NOs:1 or 3, corresponds to a naturally-occurring nucleic acidmolecule.

[0137] As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein).

[0138] As used herein, the terms “gene” and “recombinant gene” refer tonucleic acid molecules that include an open reading frame encoding a18232 protein, preferably a mammalian 18232 protein, and further caninclude non-coding regulatory sequences and introns.

[0139] An “isolated” or “purified” polypeptide or protein issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived,.orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. In one embodiment, the language “substantiallyfree” means preparation of 18232 protein having less than about 30%,20%, 10% and more preferably 5% (by dry weight), of non-18232 protein(also referred to herein as a “contaminating protein”), or of chemicalprecursors or non-18232 chemicals. When the 18232 protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The invention includes isolated or purified preparations ofat least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0140] A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of 18232 (e.g., the sequence of SEQID NO:1 or 3, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number ______) withoutabolishing or more preferably, without substantially altering abiological activity of the 18232 protein, whereas an “essential” aminoacid residue results in such a change. For example, amino acid residuesthat are conserved among the polypeptides of the present invention,e.g., those present in the dual specificity phosphatase catalyticdomain, are predicted to be particularly unamenable to alteration.

[0141] A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a 18232protein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a 18232 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for 18232 biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO:1 or 3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number ______, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

[0142] As used herein, a “biologically active portion” of a 18232protein includes a fragment of a 18232 protein that participates in aninteraction between a 18232 molecule and a non-18232 molecule.Biologically active portions of a 18232 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the 18232 protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include less amino acids than thefull length 18232 protein and exhibit at least one activity of a 18232protein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the 18232 protein, e.g., dualspecificity phosphatase activity. A biologically active portion of a18232 protein can be a polypeptide that is, for example, 10, 25, 50,100, 150 or more amino acids in length. Biologically active portions ofa 18232 protein can be used as targets for developing agents thatmodulate a 18232 mediated activity, e.g., dual specificity phosphataseactivity.

[0143] Particular 18232 polypeptides of the present invention have anamino acid sequence sufficiently identical to the amino acid sequence ofSEQ ID NO:2. The term “sufficiently identical” or “substantiallyidentical” is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient or minimum number of identical orequivalent (e.g., with a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences have a commonstructural domain or common functional activity. For example, amino acidor nucleotide sequences that contain a common structural domain havingat least about 60%, or 65% identity, likely 75% identity, more likely85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity aredefined herein as sufficiently or substantially identical.

[0144] Calculations of homology or sequence identity between sequences(the terms are used interchangeably herein) are performed as follows. Todetermine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence (e.g., when aligning a second sequence to the 18232amino acid sequence of SEQ ID NO:2 having 59 amino acid residues, atleast 70, preferably at least 88, more preferably at least 106, evenmore preferably at least 123, and even more preferably at least 141,158, or 176 amino acid residues are aligned). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

[0145] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within the invention) is using aBlossum 62 scoring matrix with a gap open penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

[0146] The percent identity between two amino acid or nucleotidesequences can be determined using the algorithm of E. Meyers and W.Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

[0147] The nucleic acid and protein sequences described herein can beused as a “query sequence” to perform a search against public databasesto, for example, identify other family members or related sequences.Such searches can be performed using the NBLAST and XBLAST programs(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 18232 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 18232 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

[0148] “Misexpression or aberrant expression”, as used herein, refers toa non-wild type pattern of gene expression at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.“Subject”, as used herein, can refer to a mammal, e.g., a human, or toan experimental animal or disease model. The subject also can be anon-human animal, e.g., a horse, cow, goat, or other domestic animal.

[0149] A “purified preparation of cells”, as used herein, refers to, inthe case of plant or animal cells, an in vitro preparation of cells andnot an entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

[0150] Various aspects of the invention are described in further detailbelow.

[0151] Isolated Nucleic Acid Molecules

[0152] In one aspect, the invention provides an isolated or purifiednucleic acid molecule that encodes a 18232 polypeptide described herein,e.g., a full length 18232 protein or a fragment thereof, e.g., abiologically active portion of a 18232 protein. Also included is anucleic acid fragment suitable for use as a hybridization probe, whichcan be used, e.g., to identify a nucleic acid molecule encoding apolypeptide of the invention, 18232 mRNA, or fragments suitable for useas primers, e.g., PCR primers for the amplification or mutation ofnucleic acid molecules.

[0153] In one embodiment, an isolated nucleic acid molecule of theinvention includes the nucleotide sequence shown in SEQ ID NO:1, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______, or a portion of any of these nucleotidesequences. In one embodiment, the nucleic acid molecule includessequences encoding the 18232 protein (i.e., “the coding region”, fromnucleotides 329-859 of SEQ ID NO:1), as well as 5′ untranslatedsequences (nucleotides 1-328 of SEQ ID NO:1). Alternatively, the nucleicacid molecule can include only the coding region of SEQ ID NO:1 (e.g.,nucleotides 329-859, corresponding to SEQ ID NO:3) and, e.g., noflanking sequences that normally accompany the subject sequence. Inanother embodiment, the nucleic acid molecule encodes a sequencecorresponding to the mature protein from about amino acid 1 to aminoacid 176 of SEQ ID NO:2.

[0154] In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule that is a complement of thenucleotide sequence shown in SEQ ID NO:1 or 3, the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number______, or a portion of any of these nucleotide sequences. In otherembodiments, the nucleic acid molecule of the invention is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO:1 or 3, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number ______ such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number ______, thereby forming a stable duplex.

[0155] In one embodiment, an isolated nucleic acid molecule of thepresent invention includes a nucleotide sequence that is at least about:60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more homologous to the entire length of the nucleotidesequence shown in SEQ ID NO:1 or 3, or the entire length of thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______. In the case of an isolated nucleic acidmolecule which is longer than or equivalent in length to the referencesequence, e.g., SEQ ID NO:1, the comparison is made with the full lengthof the reference sequence. Where the isolated nucleic acid molecule isshorter that the reference sequence, e.g., shorter than SEQ ID NO:1, thecomparison is made to a segment of the reference sequence of the samelength (excluding any loop required by the homology calculation).

[0156] 18232 Nucleic Acid Fragments

[0157] A nucleic acid molecule of the invention can include only aportion of the nucleic acid sequence of SEQ ID NO:1 or 3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______. For example, such a nucleic acid moleculecan include a fragment that can be used as a probe or primer or afragment encoding a portion of a 18232 protein, e.g., an immunogenic orbiologically active portion of a 18232 protein. A fragment can comprisenucleotides 380 to 796 of SEQ ID NO:1, which encodes a dual specificityphosphatase catalytic domain of human 18232. The nucleotide sequencedetermined from the cloning of the 18232 gene allows for the generationof probes and primers designed for use in identifying and/or cloningother 18232 family members, or fragments thereof, as well as 18232homologues or fragments thereof, from other species.

[0158] In another embodiment, a nucleic acid includes a nucleotidesequence that includes part, or all, of the coding region and extendsinto either (or both) the 5′ or 3′ noncoding region. Other embodimentsinclude a fragment that includes a nucleotide sequence encoding an aminoacid fragment described herein. Nucleic acid fragments can encode aspecific domain or site described herein or fragments thereof,particularly fragments thereof which are at least 400 amino acids inlength. Fragments also include nucleic acid sequences corresponding tospecific amino acid sequences described above or fragments thereof.Nucleic acid fragments should not to be construed as encompassing thosefragments that may have been disclosed prior to the invention.

[0159] A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment also can include one or more domains, regions, or functionalsites described herein. Thus, for example, the nucleic acid fragment caninclude a dual specificity phosphatase catalytic domain and an amidationsite.

[0160] 18232 probes and primers are provided. Typically a probe/primeris an isolated or purified oligonucleotide. The oligonucleotidetypically includes a region of nucleotide sequence that hybridizes understringent conditions to at least about 7, 12 or 15, preferably about 20or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense or antisense sequence of SEQ ID NO:1or 3, the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC as Accession Number ______, of a naturally occurring allelicvariant or mutant of SEQ ID NO:1 or 3, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number______.

[0161] In a preferred embodiment the nucleic acid is a probe that is atleast 5 or 10 and less than 500, 300, or 200 base pains in length, andmore preferably is less than 100 or less than 50 base pairs in length.It should be identical, or differ by 1, or less than 5 or 10 bases, froma sequence disclosed herein. If alignment is needed for this comparison,the sequences should be aligned for maximum homology. “Looped” outsequences in the alignment from deletions, insertions, or mismatches,are considered differences.

[0162] A probe or primer can be derived from the sense or anti-sensestrand of a nucleic acid that encodes a dual specificity phosphatasecatalytic domain: amino acid residues 18 to 156 of SEQ ID NO:2.

[0163] In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 18232 sequence, e.g., one of the ones described herein. Theprimers should be at least 5, 10, or 50 base pairs in length and lessthan 100 or 200 base pairs in length. The primers should be identical,or differ by one base from a sequence disclosed herein or from anaturally occurring variant. E.g., primers suitable for amplifying allor a portion of the dual specificity phosphatase catalytic domain (aminoacid residues 18 to 156 of SEQ ID NO:2).

[0164] A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

[0165] A nucleic acid fragment encoding a “biologically active portionof a 18232 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1 or 3, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number______, which encodes a polypeptide having a 18232 biological activity(e.g., the biological activities of the 18232 proteins describedherein), expressing the encoded portion of the 18232 protein (e.g., byrecombinant expression in vitro) and assessing the activity of theencoded portion of the 18232 protein. For example, a nucleic acidfragment encoding a biologically active portion of 18232 includes a dualspecificity phosphatase catalytic domain, e.g., amino acid residues 18to 156 of SEQ ID NO:2. A nucleic acid fragment encoding a biologicallyactive portion of a 18232 polypeptide, may comprise a nucleotidesequence that is greater than about 300 or more nucleotides in length(e.g., greater than about 400 nucleotides in length).

[0166] In a preferred embodiment, the fragment is at least 381, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000nucleotides in length, or more nucleotides in length and hybridizesunder stringent hybridization conditions to a nucleic acid molecule ofSEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number ______.

[0167] In a preferred embodiment, a nucleic acid fragment includes anucleotide sequence comprising nucleotides SEQ ID NO:1 or SEQ ID NO:3,or a portion thereof, wherein each portion is about 381 or longernucleotides and hybridizes under stringent hybridization conditions to anucleic acid molecule of SEQ ID NO:1, or SEQ ID NO:3, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number ______.

[0168] In a preferred embodiment, a nucleic acid fragment has anucleotide sequence other than (e.g., differs by at least one, two,three, five, ten or more nucleotides from) the nucleotide sequence ofsequence of AI 018626, AW 206269, AI 672432, AI 034374, AA 700744, AI027213, AI 498377, AI 950221, AI 016945, or AI 040185.

[0169] 18232 Nucleic Acid Variants

[0170] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO:1 or 3, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______. Such differences can be due to degeneracy ofthe genetic code (and result in a nucleic acid that encodes the same18232 proteins as those encoded by the nucleotide sequence disclosedherein. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence that differs by at least 1, but less than 5, 10, 20, 50,or 100 amino acid residues than that shown in SEQ ID NO:2. If alignmentis needed for this comparison the sequences should be aligned formaximum homology. “Looped” out sequences from deletions, insertions, ormismatches, are considered differences.

[0171] Nucleic acids of the invention can be chosen for having codons,which are preferred, or non-preferred, for a particular expressionsystem. E.g., the nucleic acid can be one in which at least one codon,at preferably at least 10%, or 20% of the codons has been altered suchthat the sequence is optimized for expression in E. coli, yeast, human,insect, or Chinese hamster ovary (CHO) cells.

[0172] Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non-naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared with the encoded product).

[0173] In a preferred embodiment, the nucleic acid differs from that ofSEQ ID NO:1 or 3, or the sequence in ATCC Accession Number ______, e.g.,as follows: by at least one but less than 10, 20, 30, or 40 nucleotides;at least one but less than 1%, 5%, 10% or 20% of the nucleotides in thesubject nucleic acid. If necessary for this analysis, the sequencesshould be aligned for maximum homology. “Looped” out sequences fromdeletions, insertions, or mismatches, are considered differences.

[0174] Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the amino acidsequence shown in SEQ ID NO:2 or a fragment of this sequence. Suchnucleic acid molecules can readily be identified as being able tohybridize under stringent conditions, to the nucleotide sequence shownin SEQ ID NO 1 or a fragment of the sequence. Nucleic acid moleculescorresponding to orthologs, homologs, and allelic variants of the 18232cDNAs of the invention can further be isolated by mapping to the samechromosome or locus as the 18232 gene. Preferred variants include thosethat are correlated with tyrosine phosphatase activity.

[0175] Allelic variants of 18232, e.g., human 18232, include bothfunctional and non-functional proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the 18232 proteinwithin a population that maintain the ability to remove the phosphatefrom a tyrosine, serine, or threonine residue of a phosphorylatedprotein. Functional allelic variants typically will contain onlyconservative substitution of one or more amino acids of SEQ ID NO:2, orsubstitution, deletion or insertion of non-critical residues innon-critical regions of the protein. Non-functional allelic variants arenaturally-occurring amino acid sequence variants of the 18232, e.g.,human 18232, protein within a population that do not have the ability toremove the phosphate from a tyrosine serine, or threonine residue of aphosphorylated protein. Non-functional allelic variants will typicallycontain a non-conservative substitution, a deletion, or insertion, orpremature truncation of the amino acid sequence of SEQ ID NO:2, or asubstitution, insertion, or deletion in critical residues or criticalregions of the protein.

[0176] Moreover, nucleic acid molecules encoding other 18232 familymembers and, thus have a nucleotide sequence that differs from the 18232sequences of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number ______ areintended to be within the scope of the invention.

[0177] Antisense Nucleic Acid Molecules, Ribozymes and Modified 18232Nucleic Acid Molecules

[0178] In another aspect, the invention features, an isolated nucleicacid molecule that is antisense to 18232. An “antisense” nucleic acidcan include a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire18232 coding strand, or to only a portion thereof (e.g., the codingregion of 18232 corresponding to SEQ ID NO:3). In another embodiment,the antisense nucleic acid molecule is antisense to a “noncoding region”of the coding strand of a nucleotide sequence encoding 18232 (e.g., the5′ and 3′ untranslated regions).

[0179] An antisense nucleic acid can be designed such that it iscomplementary to the entire coding region of 18232 mRNA, but morepreferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of 18232 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 18232 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0180] An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions withprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. The antisense nucleicacid also can be produced biologically using an expression vector intowhich a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest, describedfurther in the following subsection).

[0181] The antisense nucleic acid molecules of the invention aretypically administered to a subject (e.g., by direct injection at atissue site), or generated in situ such that they hybridize with or bindto cellular mRNA and/or genomic DNA encoding a 18232 protein to therebyinhibit expression of the protein, e.g., by inhibiting transcriptionand/or translation. Alternatively, antisense nucleic acid molecules canbe modified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically bind to receptors or antigens expressed on aselected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies that bind to cell surface receptorsor antigens. The antisense nucleic acid molecules can also be deliveredto cells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong polymerase II or polymerase III promoter arepreferred.

[0182] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0183] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. A ribozyme having specificity for a18232-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a 18232 cDNA disclosedherein (i.e., SEQ ID NO:1 or 3), and a sequence having known catalyticsequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 orHaselhoff and Gerlach (1988) Nature 334:585-591). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a 18232-encoding mRNA. See, e.g.,Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, 18232 mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

[0184] 18232 gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of the 18232 (e.g., the18232 promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 18232 gene in target cells. See generally,Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al.(1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15. The potential sequences that can be targeted for triplehelix formation can be increased by creating a “switchback” nucleic acidmolecule. Switchback molecules are synthesized in an alternating 5′-3′,3′-5′ manner, such that they base pair with first one strand of a duplexand then the other, eliminating the necessity for a sizeable stretch ofeither purines or pyrimidines to be present on one strand of a duplex.

[0185] The invention also provides detectably labeled oligonucleotideprimer and probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

[0186] A 18232 nucleic acid molecule can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., aDNA mimic in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength. Thesynthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described in Hyrup B. et al. (1996)supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0187] PNAs of 18232 nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of 18232 nucleic acid molecules can also beused in the analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup B.(1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0188] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier(see, e.g., PCT Publication No. WO89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

[0189] The invention also includes molecular beacon oligonucleotideprimer and probe molecules having at least one region that iscomplementary to a 18232 nucleic acid of the invention. The molecularbeacon primer and probe molecules also have two complementary regions,one having a fluorophore and one having a quencher, such that themolecular beacon is useful for quantitating the presence of a 18232nucleic acid of the invention in a sample. Molecular beacon nucleicacids are described, for example, in Lizardi et al., U.S. Pat. No.5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al.,U.S. Pat. No. 5,876,930.

[0190] Isolated 18232 Polypeptides

[0191] In another aspect, the invention features an isolated 18232protein or fragment thereof, e.g., a biologically active portion for useas immunogens or antigens to raise or test (or more generally to bind)anti-18232 antibodies. 18232 protein can be isolated from cells ortissue sources using standard protein purification techniques. 18232protein or fragments thereof can be produced by recombinant DNAtechniques or synthesized chemically.

[0192] Polypeptides of the invention include those that arise as aresult of the existence of multiple genes, alternative transcriptionevents, alternative RNA splicing events, and alternative translationaland posttranslational events. The polypeptide can be expressed insystems, e.g., cultured cells, which result in substantially the sameposttranslational modifications present when expressed the polypeptideis expressed in a native cell, or in systems which result in thealteration or omission of posttranslational modifications, e.g.,glycosylation or cleavage, present when expressed in a native cell.

[0193] In a preferred embodiment, a 18232 polypeptide has one or more ofthe following characteristics:

[0194] (i) it has the ability to promote removal of phosphate fromphosphorylated tyrosine, serine, or threonine residues of protein;

[0195] (ii) it has a molecular weight (e.g., a deduced molecularweight), amino acid composition or other physical characteristic of a18232 protein of SEQ ID NO:2;

[0196] (iii) it has an overall sequence similarity of at least 60%, morepreferably at least 70, 80, 90, or 95%, with a polypeptide encoded bySEQ ID NO:2;

[0197] (iv) it has a phosphatase catalytic domain which is preferablyabout 70%, 80%, 90% or 95% with amino acid residues 18-156 of SEQ IDNO:2; or

[0198] (v) it has at least 70%, preferably at least 80%, and mostpreferably at least 95% of the cysteines found in the amino acidsequence of the native protein.

[0199] In a preferred embodiment, the 18232 protein or fragment thereofdiffers from the corresponding sequence in SEQ ID NO:2. In oneembodiment, it differs by at least one but by less than 15, 10 or 5amino acid residues. In another embodiment, it differs from thecorresponding sequence in SEQ ID NO:2 by at least one residue but lessthan 20%, 15%, 10% or 5% of the residues in it differ from thecorresponding sequence in SEQ ID NO:2. (If this comparison requiresalignment, the sequences should be aligned for maximum homology.“Looped” out sequences from deletions, insertions, or mismatches, areconsidered differences.) The differences are, preferably, differences orchanges at a non-essential residue or a conservative substitution. In apreferred embodiment, the differences are not in the dual specificityphosphatase catalytic domain. In another preferred embodiment one ormore differences are at non-active site residues, e.g., amino acids1-17, or 157-176 of SEQ ID NO:2.

[0200] Other embodiments include a protein that contains one or morechanges in amino acid sequence, e.g., a change in an amino acid residuethat is not essential for activity. Such 18232 proteins differ in aminoacid sequence from SEQ ID NO:2, yet retain biological activity.

[0201] In one embodiment, the protein includes an amino acid sequence atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to SEQ ID NO:2.

[0202] A 18232 protein or fragment is provided which varies from thesequence of SEQ ID NO:2 in non-active site residues by at least one butby less than 15, 10 or 5 amino acid residues in the protein or fragment,but which does not differ from SEQ ID NO:2 in regions having phosphatasecatalytic activity. (If this comparison requires alignment the sequencesshould be aligned for maximum homology. “Looped” out sequences fromdeletions, insertions, or mismatches, are considered differences.) Insome embodiments, the difference is at a non essential residue or is aconservative substitution, while in others, the difference is at anessential residue or is a non conservative substitution.

[0203] In one embodiment, a biologically active portion of a 18232protein includes a dual specificity phosphatase catalytic domain.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native 18232protein.

[0204] In a preferred embodiment, the 18232 protein has an amino acidsequence shown in SEQ ID NO:2. In other embodiments, the 18232 proteinis substantially identical to SEQ ID NO:2. In yet another embodiment,the 18232 protein is substantially identical to SEQ ID NO:2 and retainsthe functional activity of the protein of SEQ ID NO:2, as described indetail in the subsections above. Accordingly, in another embodiment, the18232 protein is a protein which includes an amino acid sequence atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or moreidentical to SEQ ID NO:2.

[0205] 18232 Chimeric or Fusion Proteins

[0206] In another aspect, the invention provides 18232 chimeric orfusion proteins. As used herein, a 18232 “chimeric protein” or “fusionprotein” includes a 18232 polypeptide linked to a non-18232 polypeptide.A “non-18232 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein that is not substantially homologousto the 18232 protein, e.g., a protein that is different from the 18232protein and that is derived from the same or a different organism. The18232 polypeptide of the fusion protein can correspond to all or aportion e.g., a fragment described herein of a 18232 amino acidsequence. In a preferred embodiment, a 18232 fusion protein includes atleast one (e.g. two) biologically active portion of a 18232 protein. Thenon-18232 polypeptide can be fused to the N-terminus or C-terminus ofthe 18232 polypeptide.

[0207] The fusion protein can include a moiety that has high affinityfor a ligand. For example, the fusion protein can be a GST-18232 fusionprotein in which the 18232 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant 18232. Alternatively, the fusion protein can be a 18232protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 18232 can be increased through use of a heterologous signalsequence.

[0208] Fusion proteins can include all or a part of a serum protein,e.g., an IgG constant region, or human serum albumin.

[0209] The 18232 fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo.The 18232 fusion proteins can be used to affect the bioavailability of a18232 substrate. 18232 fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 18232 protein; (ii)mis-regulation of the 18232 gene; and (iii) aberrant post-translationalmodification of a 18232 protein.

[0210] Moreover, 18232-fusion proteins of the invention can be used asimmunogens to produce anti-18232 antibodies in a subject, to purify18232 ligands, and in screening assays to identify molecules thatinhibit the interaction of 18232 with a 18232 substrate.

[0211] Expression vectors are commercially available that already encodea fusion moiety (e.g., a GST polypeptide). A 18232-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 18232 protein.

[0212] Variants of 18232 Proteins

[0213] In another aspect, the invention features a variant of a 18232polypeptide, e.g., a polypeptide that functions as an agonist (mimetic)or as an antagonist of 18232 activities. Variants of the 18232 proteinscan be generated by mutagenesis, e.g., discrete point mutations, theinsertion or deletion of sequences or the truncation of a 18232 protein.An agonist of the 18232 protein retains substantially the same, or asubset, of the biological activities of the naturally occurring form ofa 18232 protein. An antagonist of a 18232 protein can inhibit one ormore of the activities of the naturally occurring form of the 18232protein by, for example, competitively modulating a 18232-mediatedactivity of a 18232 protein. Thus, specific biological effects can beelicited by treatment with a variant of limited function. Preferably,treatment of a subject with a variant having a subset of the biologicalactivities of the naturally occurring form of the protein has fewer sideeffects in a subject relative to treatment with the naturally occurringform of the 18232 protein.

[0214] Variants of a 18232 protein can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a 18232protein for agonist or antagonist activity.

[0215] Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 18232 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 18232 protein.

[0216] Variants in which a cysteine residue is added or deleted or inwhich a residue that is glycosylated is added or deleted areparticularly preferred.

[0217] Methods for screening gene products of combinatorial librariesmade by point mutations or truncation, and for screening cDNA librariesfor gene products having a selected property are known. Recursiveensemble mutagenesis (REM), a new technique which enhances the frequencyof functional mutants in the libraries, can be used in combination withscreening assays to identify 18232 variants (Arkin and Yourvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) ProteinEngineering 6(3):327-331).

[0218] Cell based assays can be exploited to analyze a variegated 18232library. For example, a library of expression vectors can be transfectedinto a cell line, e.g., a cell line, which ordinarily responds to 18232in a substrate-dependent manner. The transfected cells are thencontacted with 18232 and the effect of the expression of the mutant onsignaling by the 18232 substrate can be detected, e.g., by measuringloss of phosphorylation of tyrosine, serine, or threonine residues.Plasmid DNA can then be recovered from the cells that score forinhibition, or alternatively, potentiation of signaling by the 18232substrate, and the individual clones further characterized.

[0219] In another aspect, the invention features a method of making a18232 polypeptide, e.g., a peptide having a non-wild type activity,e.g., an antagonist, agonist, or super agonist of a naturally occurring18232 polypeptide, e.g., a naturally occurring 18232 polypeptide. Themethod includes: altering the sequence of a 18232 polypeptide, e.g., bysubstitution or deletion of one or more residues of a non-conservedregion, a domain ,or residue disclosed herein, and testing the alteredpolypeptide for the desired activity.

[0220] In another aspect, the invention features a method of making afragment or analog of a 18232 polypeptide that retains at least onebiological activity of a naturally occurring 18232 polypeptide. Themethod includes: altering the sequence, e.g., by substitution ordeletion of one or more residues, of a 18232 polypeptide, e.g., alteringthe sequence of a non-conserved region, or a domain or residue describedherein, and testing the altered polypeptide for the desired activity.

[0221] Anti-18232 Antibodies

[0222] In another aspect, the invention provides an anti-18232 antibody.The term “antibody” as used herein refers to an immunoglobulin moleculeor immunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include F(ab) and F(ab′)₂ fragments that can be generated bytreating the antibody with an enzyme such as pepsin.

[0223] The antibody can be a polyclonal, monoclonal, recombinant, e.g.,a chimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

[0224] A full-length 18232 protein, or antigenic peptide fragment of18232, can be used as an immunogen or can be used to identify anti-18232antibodies made with other immunogens, e.g., cells, membranepreparations, and the like. The antigenic peptide of 18232 shouldinclude at least 8 amino acid residues of the amino acid sequence shownin SEQ ID NO:2 and encompasses an epitope of 18232. Preferably, theantigenic peptide includes at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

[0225] Fragments of 18232 that include residues 51-61, 101-111, or131-155 of SEQ ID NO:2 can be used to make, e.g., used as immunogens orused to characterize the specificity of an antibody, antibodies againstregions of the 18232 protein which are believed to be hydrophilic.Similarly, a fragment of 18232 that includes residues 11-31, 38-51, or81-101 of SEQ ID NO:2 can be used to make an antibody against regions ofthe 18232 protein which are believed to be hydrophobic; a fragment of18232 that includes residues 18-28, 38-68, or 128-156 of SEQ ID NO:2 canbe used to make an antibody against the dual specificity phosphatasecatalytic region of the 18232 protein.

[0226] Antibodies reactive with, or specific for, any of these regions,or other regions or domains described herein are provided.

[0227] Preferred epitopes encompassed by the antigenic peptide areregions of 18232 that are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity. Forexample, an Emini surface probability analysis of the human 18232protein sequence can be used to indicate the regions that have aparticularly high probability of being localized to the surface of the18232 protein and are thus likely to constitute surface residues usefulfor targeting antibody production.

[0228] In a preferred embodiment the antibody binds an epitope on anydomain or region on 18232 proteins described herein.

[0229] Chimeric, humanized, but most preferably, completely humanantibodies are desirable for applications which include repeatedadministration, e.g., therapeutic treatment (and some diagnosticapplications) of human patients.

[0230] The anti-18232 antibody can be a single chain antibody. Asingle-chain antibody (scFV) may be engineered (see, for example,Colcher, D., et al. (1999) Ann N Y Acad Sci Jun 30;880:263-80; andReiter, Y. (1996) Clin Cancer Res, Feb;2(2):245-52). The single chainantibody can be dimerized or multimerized to generate multivalentantibodies having specificities for different epitopes of the sametarget 18232 protein.

[0231] An anti-18232 antibody (e.g., monoclonal antibody) can be used toisolate 18232 by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an anti-18232 antibody can be used todetect 18232 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-18232 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance (i.e., antibody labelling). Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

[0232] Recombinant Expression Vectors, Host Cells and GeneticallyEngineered Cells

[0233] In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

[0234] A vector can include a 18232 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably, therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those that direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 18232 proteins,mutant forms of 18232 proteins, fusion proteins, and the like).

[0235] The recombinant expression vectors of the invention can bedesigned for expression of 18232 proteins in prokaryotic or eukaryoticcells. For example, polypeptides of the invention can be expressed in E.coli, insect cells (e.g., using baculovirus expression vectors), yeastcells or mammalian cells. Suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0236] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase(GST), maltose E binding protein, and protein A, respectively, to thetarget recombinant protein.

[0237] Purified fusion proteins can be used in 18232 activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for 18232 proteins. In a preferredembodiment, a fusion protein expressed in a retroviral expression vectorof the present invention can be used to infect bone marrow cells thatare subsequently transplanted into irradiated recipients. The pathologyof the subject recipient is then examined after sufficient time haspassed (e.g., six weeks).

[0238] To maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

[0239] The 18232 expression vector can be a yeast expression vector, avector for expression in insect cells, e.g., a baculovirus expressionvector or a vector suitable for expression in mammalian cells.

[0240] When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40.

[0241] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid). Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, including forexample, the murine hox promoters (Kessel and Gruss (1990) Science249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989)Genes Dev. 3:537-546).

[0242] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub, H. et al., Antisense RNA as amolecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986.

[0243] Another aspect the invention provides a host cell that includes anucleic acid molecule described herein, e.g., a 18232 nucleic acidmolecule within a recombinant expression vector or a 18232 nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0244] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a 18232 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as CHO or COS cells).Other suitable host cells are known to those skilled in the art.

[0245] Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation

[0246] A host cell of the invention can be used to produce (i.e.,express) a 18232 protein. Accordingly, the invention further providesmethods for producing a 18232 protein using the host cells of theinvention. In one embodiment, the method includes culturing the hostcell of the invention (into which a recombinant expression vectorencoding a 18232 protein has been introduced) in a suitable medium suchthat a 18232 protein is produced. In another embodiment, the methodfurther includes isolating a 18232 protein from the medium or the hostcell.

[0247] In another aspect, the invention features a cell or purifiedpreparation of cells that include a 18232 transgene, or which otherwisemisexpress 18232. The cell preparation can consist of human or non-humancells, e.g., rodent cells such as mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a 18232transgene, e.g., a heterologous form of a 18232 nucleic acid, e.g., agene derived from humans (in the case of a non-human cell). The 18232transgene can be misexpressed, e.g., overexpressed or underexpressed. Inother preferred embodiments, the cell or cells include a gene thatmisexpresses an endogenous 18232 nucleic acid, e.g., disruption in theexpression of a gene, e.g., a knockout. Such cells can serve as a modelfor studying disorders that are related to mutated or mis-expressed18232 alleles or for use in drug screening.

[0248] In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid that encodes a18232 polypeptide.

[0249] Also provided are cells (e.g., human cells, e.g., a hematopoieticcell or a fibroblast cell), or a purified preparation thereof, in whichan endogenous 18232 is under the control of a regulatory sequence thatdoes not normally control the expression of the endogenous 18232 gene.The expression characteristics of an endogenous gene within a cell,e.g., a cell line or microorganism, can be modified by inserting aheterologous DNA regulatory element into the genome of the cell suchthat the inserted regulatory element is operably linked to theendogenous 18232 gene. For example, an endogenous 18232 gene, e.g., agene which is “transcriptionally silent,” e.g., not normally expressed,or expressed only at very low levels, may be activated by inserting aregulatory element which is capable of promoting the expression of anormally expressed gene product in that cell. Techniques such astargeted homologous recombinations, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published in May 16, 1991.

[0250] Transgenic Animals

[0251] The invention provides non-human transgenic animals. Such animalsare useful for studying the function and/or activity of a 18232 proteinand for identifying and/or evaluating modulators of 18232 activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangement, e.g., a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 18232 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

[0252] Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a transgene of the invention to direct expression of a 18232protein to particular cells. A transgenic founder animal can beidentified based upon the presence of a 18232 transgene in its genomeand/or expression of 18232 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a 18232 protein can further be bred to othertransgenic animals carrying other transgenes.

[0253] 18232 proteins or polypeptides can be expressed in transgenicanimals or plants, e.g., a nucleic acid encoding the protein orpolypeptide can be introduced into the genome of an animal. In preferredembodiments the nucleic acid is placed under the control of a tissuespecific promoter, e.g., a milk or egg specific promoter, and recoveredfrom the milk or eggs produced by the animal. Suitable animals are mice,pigs, cows, goats, and sheep.

[0254] The invention also includes a population of cells from atransgenic animal, as discussed herein.

[0255] Uses

[0256] The nucleic acid molecules, proteins, protein homologues, andantibodies described herein can be used in one or more of the followingmethods: a) screening assays; b) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenetics); and c) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used, for example, to express a 18232 protein (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect a 18232 mRNA (e.g., in a biological sample) ora genetic alteration in a 18232 gene, and to modulate 18232 activity, asdescribed further below. The 18232 proteins can be used to treatdisorders characterized by insufficient or excessive production of a18232 substrate or production of 18232 inhibitors. In addition, the18232 proteins can be used to screen for naturally occurring 18232substrates, to screen for drugs or compounds that modulate 18232activity, as well as to treat disorders characterized by insufficient orexcessive production of 18232 protein or production of 18232 proteinforms which have decreased, aberrant or unwanted activity compared to18232 wild type protein (e.g., imbalance of protein tyrosine kinase andprotein tyrosine phosphorylase activities, leading to aberrantproliferation and/or differentiation of hematopoietic cells). Moreover,the anti-18232 antibodies of the invention can be used to detect andisolate 18232 proteins, regulate the bioavailability of 18232 proteins,and modulate 18232 activity.

[0257] A method of evaluating a compound for the ability to interactwith, e.g., bind, a subject 18232 polypeptide is provided. The methodincludes: contacting the compound with the subject 18232 polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with the subject 18232 polypeptide. This method can beperformed in vitro, e.g., in a cell free system, or in vivo, e.g., in atwo-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules that interact with a subject 18232polypeptide. It can also be used to find natural or synthetic inhibitorsof a subject 18232 polypeptide. Screening methods are discussed in moredetail below.

[0258] Screening Assays:

[0259] The invention provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs) that bind to 18232 proteins,have a stimulatory or inhibitory effect on, for example, 18232expression or 18232 activity, or have a stimulatory or inhibitory effecton, for example, the expression or activity of a 18232 substrate.Compounds thus identified can be used to modulate the activity of targetgene products (e.g., 18232 genes) in a therapeutic protocol, toelaborate the biological function of the target gene product, or toidentify compounds that disrupt normal target gene interactions.

[0260] In one embodiment, the invention provides assays for screeningcandidate or test compounds that are substrates of a 18232 protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds that bind to or modulate the activity of a 18232 proteinor polypeptide or a biologically active portion thereof.

[0261] In any screening assay, a 18232 polypeptide which may have, e.g.,a dual specificity domain, can be used.

[0262] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; peptoid libraries [librariesof molecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive] (see, e.g., Zuckermann, R. N. etal. J. Med Chem. 1994, 37: 2678-85); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library and peptoid library approaches are limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

[0263] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0264] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria orspores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992)Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990)Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla etal. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. MolBiol. 222:301-310); (Ladner supra.).

[0265] In one embodiment, an assay is a cell-based assay in which a cellthat expresses a 18232 protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate 18232 activity is determined. Determining the ability of thetest compound to modulate 18232 activity can be accomplished bymonitoring, for example, phosphatase activity. The cell, for example,can be of mammalian origin, e.g., human.

[0266] The ability of the test compound to modulate 18232 binding to acompound, e.g., a 18232 substrate, or to bind to 18232 can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to 18232 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, 18232 can be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulate18232 binding to a 18232 substrate in a complex. For example, compounds(e.g., 18232 substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

[0267] The ability of a compound (e.g., a 18232 substrate) to interactwith 18232 with or without the labeling of any of the interactants canbe evaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 18232 without the labeling of either thecompound or 18232. McConnell, H. M. et al. (1992) Science 257:1906-1912.As used herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and 18232.

[0268] In yet another embodiment, a cell-free assay is provided in whicha 18232 protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to bind to the18232 protein or biologically active portion thereof is evaluated.Preferred biologically active portions of the 18232 proteins to be usedin assays of the present invention include fragments that participate ininteractions with non-18232 molecules, e.g., fragments with high surfaceprobability scores.

[0269] Soluble and/or membrane-bound forms of isolated proteins (e.g.,18232 proteins or biologically active portions thereof) can be used inthe cell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0270] Cell-free assays involve preparing a reaction mixture of thetarget gene protein and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected.

[0271] Assay where ability of agent to block binding of the phosphataseto the phosphorylated substrate within a cell is evaluated.

[0272] The interaction between two molecules can also be detected, e.g.,using fluorescence energy transfer (FET) (see, for example, Lakowicz etal., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

[0273] In another embodiment, determining the ability of the 18232protein to bind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalthat can be used as an indication of real-time reactions betweenbiological molecules.

[0274] In one embodiment, the target gene product or the test substanceis anchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

[0275] It may be desirable to immobilize either 18232, an anti 18232antibody or its target molecule to facilitate separation of complexedfrom uncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a18232 protein, or interaction of a 18232 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/18232 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or 18232 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of 18232binding or activity determined using standard techniques.

[0276] Other techniques for immobilizing either a 18232 protein or atarget molecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 18232 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

[0277] In order to conduct the assay, the non-immobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the immobilized component(the antibody, in turn, can be directly labeled or indirectly labeledwith, e.g., a labeled anti-Ig antibody).

[0278] In one embodiment, this assay is performed utilizing antibodiesreactive with 18232 protein or target molecules but which do notinterfere with binding of the 18232 protein to its target molecule. Suchantibodies can be derivatized to the wells of the plate, and unboundtarget or 18232 protein is trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the 18232 protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the 18232 protein or target molecule.

[0279] Alternatively, cell free assays can be conducted in a liquidphase. In such an assay, the reaction products are separated fromunreacted components by any of a number of standard techniques,including but not limited to: differential centrifugation (see, forexample, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci Aug;18(8):284-7); chromatography (gel filtration chromatography,ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. etal., eds. Current Protocols in Molecular Biology 1999, J. Wiley: NewYork.); and immunoprecipitation (see, for example, Ausubel, F. et al.,eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York).Such resins and chromatographic techniques are known to one skilled inthe art (see, e.g., Heegaard, N.H., (1998) J Mol Recognit Winter,11(1-6):141-8; Hage, D. S., and Tweed, S. A. (1997) J Chromatogr BBiomed Sci Appl Oct 10;699(1-2):499-525). Further, fluorescence energytransfer may also be conveniently utilized, as described herein, todetect binding without further purification of the complex fromsolution.

[0280] In a preferred embodiment, the assay includes contacting the18232 protein or biologically active portion thereof with a knowncompound which binds 18232 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a 18232 protein, wherein determining theability of the test compound to interact with a 18232 protein includesdetermining the ability of the test compound to preferentially bind to18232 or biologically active portion thereof, or to modulate theactivity of a target molecule, as compared to the known compound.

[0281] The target gene products of the invention can, in vivo, interactwith one or more cellular or extracellular macromolecules, such asproteins. For the purposes of this discussion, such cellular andextracellular macromolecules are referred to herein as “bindingpartners.” Compounds that disrupt such interactions can be useful inregulating the activity of the target gene product. Such compounds caninclude, but are not limited to molecules such as antibodies, peptides,and small molecules. The preferred target genes/products for use in thisembodiment are the 18232 genes herein identified. In an alternativeembodiment, the invention provides methods for determining the abilityof the test compound to modulate the activity of a 18232 protein throughmodulation of the activity of a downstream effector of a 18232 targetmolecule. For example, the activity of the effector molecule on anappropriate target can be determined, or the binding of the effector toan appropriate target can be determined, as previously described.

[0282] To identify compounds that interfere with the interaction betweenthe target gene product and its cellular or extracellular bindingpartner(s), e.g., a substrate, a reaction mixture containing the targetgene product and the binding partner is prepared, under conditions andfor a time sufficient, to allow the two products to form complex. Inorder to test an inhibitory agent, the reaction mixture is provided inthe presence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of the target gene and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the target gene product and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the target gene product and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal target gene product canalso be compared to complex formation within reaction mixturescontaining the test compound and mutant target gene product. Thiscomparison can be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normaltarget gene products.

[0283] These assays can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the target geneproduct or the binding partner onto a solid phase, and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants can bevaried to obtain different information about the compounds being tested.For example, test compounds that interfere with the interaction betweenthe target gene products and the binding partners, e.g., by competition,can be identified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

[0284] In a heterogeneous assay system, either the target gene productor the interactive cellular or extracellular binding partners, isanchored onto a solid surface (e.g., a microtiter plate), while thenon-anchored species is labeled either directly or indirectly. Theanchored species can be immobilized by non-covalent or covalentattachments. Alternatively, an immobilized antibody specific for thespecies to be anchored can be used to anchor the species to the solidsurface.

[0285] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes that have formed remainimmobilized on the solid surface. In assays where the non-immobilizedspecies is pre-labeled, the detection of label immobilized on thesurface indicates that complexes were formed. In assays where thenon-immobilized species is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the initially non-immobilized species (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody). Depending upon the order of additionof reaction components, test compounds that inhibit complex formation orthat disrupt preformed complexes can be detected.

[0286] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound. Reaction products areseparated from unreacted components and complexes detected using, forexample, an immobilized antibody specific for one of the bindingcomponents to anchor any complexes formed in solution and a labeledantibody specific for the other partner to detect anchored complexes.Again, depending upon the order of addition of reactants to the liquidphase, test compounds that inhibit complex formation or that disruptpreformed complexes can be identified.

[0287] In an alternate embodiment of the invention, a homogeneous assaycan be used. For example, a preformed complex of the target gene productand the interactive cellular or extracellular binding partner product isprepared in which either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

[0288] In yet another aspect, the 18232 proteins can be used as “baitproteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with 18232 (“18232-binding proteins” or “18232-bp”) and areinvolved in 18232 activity. Such 18232-bps can be activators orinhibitors of signals by the 18232 proteins or 18232 targets as, forexample, downstream elements of a 18232-mediated signaling pathway.

[0289] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 18232 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence from alibrary of DNA sequences that encodes an unidentified protein (“prey” or“sample”) is fused to a gene that codes for the activation domain of theknown transcription factor. (Alternatively the 18232 protein can befused to the activator domain.) If the “bait” and the “prey” proteinsare able to interact in vivo and form a 18232-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) that is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene that encodes the protein that interacts with the 18232protein.

[0290] In another embodiment, modulators of 18232 expression areidentified. For example, a cell or cell free mixture is contacted with acandidate compound and the expression of 18232 mRNA or protein evaluatedrelative to the level of expression of 18232 mRNA or protein in theabsence of the candidate compound. When expression of 18232 mRNA orprotein is greater in the presence of the candidate compound than in itsabsence, the candidate compound is identified as a stimulator of 18232mRNA or protein expression. Alternatively, when expression of 18232 mRNAor protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 18232 mRNA or protein expression. Thelevel of 18232 mRNA or protein expression can be determined by methodsdescribed herein for detecting 18232 mRNA or protein.

[0291] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulating agentcan be identified using a cell-based or a cell free assay, and theability of the agent to modulate the activity of a 18232 protein can beconfirmed in vivo, e.g., in an animal such as an animal modeloverexpressing an oncogene encoding a protein tyrosine kinase.

[0292] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 18232 modulating agent, an antisense 18232 nucleic acidmolecule, a 18232-specific antibody, or a 18232-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

[0293] Detection Assays

[0294] Portions or fragments of the nucleic acid sequences identifiedherein can be used as polynucleotide reagents. For example, thesesequences can be used to: (i) map their respective genes on a chromosomee.g., to locate gene regions associated with genetic disease or toassociate 18232 with a disease; (ii) identify an individual from aminute biological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

[0295] Chromosome Mapping

[0296] The 18232 nucleotide sequences or portions thereof can be used tomap the location of the 18232 genes on a chromosome. This process iscalled chromosome mapping. Chromosome mapping is useful in correlatingthe 18232 sequences with genes associated with disease.

[0297] Briefly, 18232 genes can be mapped to chromosomes by preparingPCR primers (preferably 15-25 bp in length) from the 18232 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 18232 sequences willyield an amplified fragment.

[0298] A panel of somatic cell hybrids in which each cell line containseither a single human chromosome or a small number of human chromosomesand a full set of mouse chromosomes, allows easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924).

[0299] Other mapping strategies e.g., in situ hybridization (describedin Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries can be used to map18232 to a chromosomal location.

[0300] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al., Human Chromosomes: A Manual of BasicTechniques (Pergamon Press, New York 1988).

[0301] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0302] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween a gene and a disease, mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland, J. etal. (1987) Nature, 325:783-787.

[0303] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the 18232 gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0304] Tissue Typing

[0305] 18232 sequences can be used to identify individuals frombiological samples using, e.g., restriction fragment length polymorphism(RFLP). In this technique, an individual's genomic DNA is digested withone or more restriction enzymes, the fragments separated, e.g., byelectrophoresis and Southern blotted, and probed to yield bands foridentification. The sequences of the present invention are useful asadditional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0306] Furthermore, the sequences of the present invention can also beused to determine the actual base-by-base DNA sequence of selectedportions of an individual's genome. Thus, the 18232 nucleotide sequencesdescribed herein can be used to prepare two PCR primers from the 5′ and3′ ends of the sequences. These primers can then be used to amplify anindividual's DNA and subsequently sequence it. Panels of correspondingDNA sequences from individuals, prepared in this manner, can provideunique individual identifications, as each individual will have a uniqueset of such DNA sequences due to allelic differences.

[0307] Allelic variation occurs to some degree in the coding regions ofthese sequences, and to a greater degree in the noncoding regions. Eachof the sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can provide positiveindividual identification with a panel of perhaps 10 to 1,000 primers,which each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3 are used, amore appropriate number of primers for positive individualidentification would be 500-2,000.

[0308] If a panel of reagents from 18232 nucleotide sequences describedherein is used to generate a unique identification database for anindividual, those same reagents can later be used to identify tissuefrom that individual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0309] Use of Partial 18232 Sequences in Forensic Biology

[0310] DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen, found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

[0311] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 (e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 and having a length of atleast 20 bases, preferably at least 30 bases) are particularlyappropriate for this use.

[0312] The 18232 nucleotide sequences described herein can further beused to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue, e.g., a tissue containing18232 phosphatase activity. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such 18232 probes can be used to identify tissue by speciesand/or by organ type.

[0313] In a similar fashion, these reagents, e.g., 18232 primers orprobes can be used to screen tissue culture for contamination (i.e.,screen for the presence of a mixture of different types of cells in aculture).

[0314] Predictive Medicine

[0315] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual.

[0316] Generally, the invention provides, a method of determining if asubject is at risk for a disorder related to a lesion in or themisexpression of a gene that encodes 18232. Such disorders include,e.g., a disorder associated with the misexpression of 18232.

[0317] The method includes one or more of the following:

[0318] detecting, in a tissue of the subject, the presence or absence ofa mutation which affects the expression of the 18232 gene, or detectingthe presence or absence of a mutation in a region which controls theexpression of the gene, e.g., a mutation in the 5′ control region;

[0319] detecting, in a tissue of the subject, the presence or absence ofa mutation which alters the structure of the 18232 gene;

[0320] detecting, in a tissue of the subject, the misexpression of the18232 gene at the mRNA level, e.g., detecting a non-wild type level of amRNA;

[0321] detecting, in a tissue of the subject, the misexpression of thegene at the protein level, e.g., detecting a non-wild type level of a18232 polypeptide.

[0322] In preferred embodiments the method includes: ascertaining theexistence of at least one of: a deletion of one or more nucleotides fromthe 18232 gene; an insertion of one or more nucleotides into the gene, apoint mutation, e.g., a substitution of one or more nucleotides of thegene, or a gross chromosomal rearrangement of the gene, e.g., atranslocation, inversion, or deletion.

[0323] For example, detecting the genetic lesion can include: (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence that hybridizes to a sense or antisensesequence from SEQ ID NO:1 or 3, or naturally occurring mutants thereofor 5′ or 3′ flanking sequences naturally associated with the 18232 gene;(ii) exposing the probe/primer to nucleic acid of the tissue; and (iii)detecting, by hybridization, e.g., in situ hybridization, of theprobe/primer to the nucleic acid, the presence or absence of the geneticlesion.

[0324] In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 18232 gene; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 18232.

[0325] Methods of the invention can be used prenatally or to determineif a subject's offspring will be at risk for a disorder.

[0326] In preferred embodiments the method includes determining thestructure of a 18232 gene, an abnormal structure being indicative ofrisk for the disorder.

[0327] In preferred embodiments the method includes contacting a sampleform the subject with an antibody to the 18232 protein or a nucleicacid, which hybridizes specifically with the gene. This and otherembodiments are discussed below.

[0328] Diagnostic and Prognostic Assays

[0329] The presence, level, or absence of 18232 protein or nucleic acidin a biological sample can be evaluated by obtaining a biological samplefrom a test subject and contacting the biological sample with a compoundor an agent capable of detecting 18232 protein or nucleic acid (e.g.,mRNA, genomic DNA) that encodes 18232 protein such that the presence of18232 protein or nucleic acid is detected in the biological sample. Theterm “biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 18232 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 18232genes; measuring the amount of protein encoded by the 18232 genes; ormeasuring the activity of the protein encoded by the 18232 genes.

[0330] The level of mRNA corresponding to the 18232 gene in a cell canbe determined both by in situ and by in vitro formats.

[0331] The isolated mRNA can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onepreferred diagnostic method for the detection of mRNA levels involvescontacting the isolated mRNA with a nucleic acid molecule (probe) thatcan hybridize to the mRNA encoded by the gene being detected. Thenucleic acid probe can be, for example, a full-length 18232 nucleicacid, such as the nucleic acid of SEQ ID NO:1, or the DNA insert of theplasmid deposited with ATCC as Accession Number ______, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to 18232 mRNA or genomic DNA. Other suitableprobes for use in the diagnostic assays are described herein.

[0332] In one format, mRNA (or cDNA) is immobilized on a surface andcontacted with the probes, for example by running the isolated mRNA onan agarose gel and transferring the mRNA from the gel to a membrane,such as nitrocellulose. In an alternative format, the probes areimmobilized on a surface and the mRNA (or cDNA) is contacted with theprobes, for example, in a two-dimensional gene chip array. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the 18232 genes.

[0333] The level of mRNA in a sample that is encoded by a 18232 nucleicacid can be evaluated with nucleic acid amplification, e.g., by rtPCR(Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques known in the art. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

[0334] For in situ methods, a cell or tissue sample can beprepared/processed and immobilized on a support, typically a glassslide, and then contacted with a probe that can hybridize to mRNA thatencodes the 18232 gene being analyzed.

[0335] In another embodiment, the methods further contacting a controlsample with a compound or agent capable of detecting 18232 mRNA, orgenomic DNA, and comparing the presence of 18232 mRNA or genomic DNA inthe control sample with the presence of 18232 mRNA or genomic DNA in thetest sample.

[0336] A variety of methods can be used to determine the level ofprotein encoded by 18232. In general, these methods include contactingan agent that selectively binds to the protein, such as an antibody witha sample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

[0337] The detection methods can be used to detect 18232 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 18232 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassays(EIA), radioimmunoassays (RIA), and Western blot analysis. In vivotechniques for detection of 18232 protein include introducing into asubject a labeled anti-18232 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

[0338] In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 18232protein, and comparing the presence of 18232 protein in the controlsample with the presence of 18232 protein in the test sample.

[0339] The invention also includes kits for detecting the presence of18232 in a biological sample. For example, the kit can include acompound or agent capable of detecting 18232 protein or mRNA in abiological sample; and a standard. The compound or agent can be packagedin a suitable container. The kit can further comprise instructions forusing the kit to detect 18232 protein or nucleic acid.

[0340] For antibody-based kits, the kit can include: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide corresponding to a marker of the invention; and, optionally,(2) a second, different antibody which binds to either the polypeptideor the first antibody and is conjugated to a detectable agent.

[0341] For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples that can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

[0342] The diagnostic methods described herein can identify subjectshaving, or at risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 18232 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

[0343] In one embodiment, a disease or disorder associated with aberrantor unwanted 18232 expression or activity is identified. A test sample isobtained from a subject and 18232 protein or nucleic acid (e.g., mRNA orgenomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 18232 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 18232 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

[0344] The prognostic assays described herein can be used to determinewhether a subject can be administered an agent (e.g., an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, smallmolecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted 18232 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a cell proliferation disorder.

[0345] The methods of the invention can also be used to detect geneticalterations in a 18232 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in18232 protein activity or nucleic acid expression, such as aproliferation disorder. In preferred embodiments, the methods includedetecting, in a sample from the subject, the presence or absence of agenetic alteration characterized by at least one of an alterationaffecting the integrity of a gene encoding a 18232 protein, or themis-expression of the 18232 gene. For example, such genetic alterationscan be detected by ascertaining the existence of at least one of 1) adeletion of one or more nucleotides from a 18232 gene; 2) an addition ofone or more nucleotides to a 18232 gene; 3) substitution of one or morenucleotides in a 18232 gene, 4) a chromosomal rearrangement of a 18232gene; 5) an alteration in the level of a messenger RNA transcript of a18232 gene, 6) aberrant modification of a 18232 gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of a 18232 gene, 8)a non-wild type level of a 18232-protein, 9) allelic loss of a 18232gene, and 10) inappropriate post-translational modification of a18232-protein.

[0346] An alteration can be detected with a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the 18232-gene. This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the sample,contacting the nucleic acid sample with one or more primers thatspecifically hybridize to a 18232 gene under conditions such thathybridization and amplification of the 18232-gene (if present) occurs,and detecting the presence or absence of an amplification product ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.Alternative amplification methods are described above and can be used ina preliminary amplification step, followed by the detection of theamplified molecules using techniques known to those of skill in the art.

[0347] In another embodiment, mutations in a 18232 gene from a samplecell can be identified by detecting alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined, e.g., by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicate mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0348] In other embodiments, genetic mutations in 18232 can beidentified by hybridizing sample and control nucleic acids, e.g., DNA orRNA, to two-dimensional arrays, e.g., chip based arrays. Such arraysinclude a plurality of addresses, each of which is positionallydistinguishable from the other. A different probe is located at eachaddress of the plurality. The arrays can have a high density ofaddresses, e.g., can contain hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, geneticmutations in 18232 can be identified in two-dimensional arrayscontaining light-generated DNA probes as described in Cronin, M. T. etal. supra. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0349] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the 18232gene and detect mutations by comparing the sequence of the sample 18232with the corresponding wild-type (control) sequence. Automatedsequencing procedures can be utilized when performing the diagnosticassays ((1995) Biotechniques 19:448), including sequencing by massspectrometry.

[0350] Other methods for detecting mutations in the 18232 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA85:4397; and Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[0351] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in 18232 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662; U.S. Pat. No. 5,459,039).

[0352] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in 18232 genes. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control 18232 nucleic acids will be denatured and allowed torenature. The secondary structure of single-stranded nucleic acidsvaries according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet 7:5).

[0353] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0354] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. NatlAcad. Sci USA 86:6230).

[0355] Alternatively, allele specific amplification technology thatdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments, amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

[0356] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga 18232 gene.

[0357] Use of 18232 Molecules as Surrogate Markers

[0358] The 18232 molecules of the invention are also useful as markersof disorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 18232 molecules of the invention may be detected,and may be correlated with one or more biological states in vivo. Forexample, the 18232 molecules of the invention may serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease may be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection may be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0359] The 18232 molecules of the invention are also useful aspharmacodynamic markers. As used herein, a “pharmacodynamic marker” isan objective biochemical marker which correlates specifically with drugeffects. The presence or quantity of a pharmacodynamic marker is notrelated to the disease state or disorder for which the drug is beingadministered; therefore, the presence or quantity of the marker isindicative of the presence or activity of the drug in a subject. Forexample, a pharmacodynamic marker may be indicative of the concentrationof the drug in a biological tissue, in that the marker is eitherexpressed or transcribed or not expressed or transcribed in that tissuein relationship to the level of the drug. In this fashion, thedistribution or uptake of the drug may be monitored by thepharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker (e.g., an 18232 marker)transcription or expression, the amplified marker may be in a quantitywhich is more readily detectable than the drug itself. Also, the markermay be more easily detected due to the nature of the marker itself; forexample, using the methods described herein, anti-18232 antibodies maybe employed in an immune-based detection system for an 18232 proteinmarker, or 18232-specific radiolabeled probes may be used to detect an18232 mRNA marker. Furthermore, the use of a pharmacodynamic marker mayoffer mechanism-based prediction of risk due to drug treatment beyondthe range of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[0360] The 18232 molecules of the invention are also useful aspharmacogenomic markers. As used herein, a “pharmacogenomic marker” isan objective biochemical marker which correlates with a specificclinical drug response or susceptibility in a subject (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence orquantity of the pharmacogenomic marker is related to the predictedresponse of the subject to a specific drug or class of drugs prior toadministration of the drug. By assessing the presence or quantity of oneor more pharmacogenomic markers in a subject, a drug therapy which ismost appropriate for the subject, or which is predicted to have agreater degree of success, may be selected. For example, based on thepresence or quantity of RNA, or protein (e.g., 18232 protein or RNA) forspecific tumor markers in a subject, a drug or course of treatment maybe selected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 18232 DNA may correlate 18232 drugresponse. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

[0361] Pharmaceutical Compositions

[0362] The nucleic acid and polypeptides, fragments thereof, as well asanti-18232 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositions. Suchcompositions typically include the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

[0363] A pharmaceutical composition is formulated to be compatible withits intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0364] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0365] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying, which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0366] Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0367] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0368] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0369] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0370] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0371] It is advantageous to formulate oral or parenteral compositionsin dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

[0372] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0373] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0374] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

[0375] For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

[0376] The present invention encompasses agents that modulate expressionor activity. An agent may, for example, be a small molecule. Forexample, such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e.,. including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

[0377] Exemplary doses include milligram or microgram amounts of thesmall molecule per kilogram of subject or sample weight (e.g., about 1μg/kg to about 500 mg/kg, about 100 μg/kg to about 5 mg/kg, or about 1μg/kg to about 50 μg/kg. It is furthermore understood that appropriatedoses of a small molecule depend upon the potency of the small moleculewith respect to the expression or activity to be modulated. When one ormore of these small molecules is to be administered to an animal (e.g.,a human) in order to modulate expression or activity of a polypeptide ornucleic acid of the invention, a physician, veterinarian, or researchermay, for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular animal subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

[0378] An antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0379] The conjugates of the invention can be used for modifying a givenbiological response. The drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

[0380] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980.

[0381] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0382] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0383] Methods of Treatment

[0384] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant or unwanted18232 expression or activity. Treatment is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease. Atherapeutic agent includes, but is not limited to, small molecules,peptides, antibodies, ribozymes and antisense oligonucleotides. Withregards to both prophylactic and therapeutic methods of treatment, suchtreatments may be specifically tailored or modified, based on knowledgeobtained from the field of pharmacogenomics as described below.

[0385] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant orunwanted 18232 expression or activity, by administering to the subject18232 or an agent that modulates 18232 expression or at least one 18232activity. Subjects at risk for a disease that is caused or contributedto by aberrant or unwanted 18232 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 18232 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of18232 aberrance, for example, a 18232 agonist or 18232 antagonist agentcan be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

[0386] It is possible that some 18232 disorders can be caused, at leastin part, by an abnormal level of gene product, or by the presence of agene product exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms. In addition to hematopoieticdisorders, 18232 disorders can include disorders of the lung, brain,heart, spinal cord, skeletal muscle, skin, and kidney, as well asnon-neoplastic immune disorders.

[0387] Examples of disorders of the lung include, but are not limitedto, congenital anomalies; atelectasis; diseases of vascular origin, suchas pulmonary congestion and edema, including hemodynamic pulmonary edemaand edema caused by microvascular injury, adult respiratory distresssyndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

[0388] Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypemephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

[0389] The 18232 nucleic acid and protein of the invention can be usedto treat and/or diagnose a variety of non-neoplastic hematopoietic orimmune disorders. Examples of hematopoieitic disorders or diseasesinclude, but are not limited to, autoimmune diseases (including, forexample, diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosis, autoimmune thyroiditis, dermatitis (including atopicdermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, and interstitial lung fibrosis), graft-versus-hostdisease, cases of transplantation, and allergy such as, atopic allergy.

[0390] Diseases of the skin, include but are not limited to, disordersof pigmentation and melanocytes, including but not limited to, vitiligo,freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, andmalignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis;disorders of epidermal maturation, including but not limited to,ichthyosis; acute inflammatory dermatoses, including but not limited to,urticaria, acute eczematous dermatitis, and erythema multiforme; chronicinflammatory dermatoses, including but not limited to, psoriasis, lichenplanus, and lupus erythematosus; blistering (bullous) diseases,including but not limited to, pemphigus, bullous pemphigoid, dermatitisherpetiformis, and noninflammatory blistering diseases: epidermolysisbullosa and porphyria; disorders of epidermal appendages, including butnot limited to, acne vulgaris; panniculitis, including but not limitedto, erythema nodosum and erythema induratum; and infection andinfestation, such as verrucae, molluscum contagiosum, impetigo,superficial fungal infections, and arthropod bites, stings, andinfestations.

[0391] Disorders involving the brain include, but are limited to,disorders involving neurons, and disorders involving glia, such asastrocytes, oligodendrocytes, ependymal cells, and microglia; cerebraledema, raised intracranial pressure and herniation, and hydrocephalus;malformations and developmental diseases, such as neural tube defects,forebrain anomalies, posterior fossa anomalies, and syringomyelia andhydromyelia; perinatal brain injury; cerebrovascular diseases, such asthose related to hypoxia, ischemia, and infarction, includinghypotension, hypoperfusion, and low-flow states—global cerebral ischemiaand focal cerebral ischemia—infarction from obstruction of local bloodsupply, intracranial hemorrhage, including intracerebral(intraparenchymal) hemorrhage, subarachnoid hemorrhage and rupturedberry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-borne (Arbo) viralencephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF 1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

[0392] Disorders involving the heart, include but are not limited to,heart failure, including but not limited to, cardiac hypertrophy,left-sided heart failure, and right-sided heart failure; ischemic heartdisease, including but not limited to angina pectoris, myocardialinfarction, chronic ischemic heart disease, and sudden cardiac death;hypertensive heart disease, including but not limited to, systemic(left-sided) hypertensive heart disease and pulmonary (right-sided)hypertensive heart disease; valvular heart disease, including but notlimited to, valvular degeneration caused by calcification, such ascalcific aortic stenosis, calcification of a congenitally bicuspidaortic valve, and mitral annular calcification, and myxomatousdegeneration of the mitral valve (mitral valve prolapse), rheumaticfever and rheumatic heart disease, infective endocarditis, andnoninfected vegetations, such as nonbacterial thrombotic endocarditisand endocarditis of systemic lupus erythematosus (Libman-Sacks disease),carcinoid heart disease, and complications of artificial valves;myocardial disease, including but not limited to dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andmyocarditis; pericardial disease, including but not limited to,pericardial effusion and hemopericardium and pericarditis, includingacute pericarditis and healed pericarditis, and rheumatoid heartdisease; neoplastic heart disease, including but not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms;congenital heart disease, including but not limited to, left-to-rightshunts—late cyanosis, such as atrial septal defect, ventricular septaldefect, patent ductus arteriosus, and atrioventricular septal defect,right-to-left shunts—early cyanosis, such as tetralogy of fallot,transposition of great arteries, truncus arteriosus, tricuspid atresia,and total anomalous pulmonary venous connection, obstructive congenitalanomalies, such as coarctation of aorta, pulmonary stenosis and atresia,and aortic stenosis and atresia, and disorders involving cardiactransplantation.

[0393] Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma; hereditary myopathies, e.g., Duchenne musculardystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy,congenital muscular dystrophy, myotonic dystrophy, facioscapulohumeralmuscular dystrophy, and oculopharyngeal dystrophy; congenitalmyopathies, e.g., central core disease, nemaline myopathy, andcentronuclear myopathy; muscular energy metabolic disorders, e.g., acidmaltase deficiency, myophosphorylase deficiency, debranching enzymedeficiency, lactate dehydrogenase deficiency, etc.; muscular lipidmetabolism disorders; e.g., myopathic carnitine deficiency, carnitinepalmityoltransferase deficiency, myodenylate deaminase deficiency;mitochondrial disorders, e.g., Kearns-Sayre syndrome, and myoclonicepilepsy and ragged red fibers syndrome; toxic myopathies; and periodicparalysis, e.g., hypokalemic periodic paralysis, hyperkalemic periodicparalysis, paramyotonia congenital, and thyrotoxic periodic paralysis.

[0394] As discussed above, successful treatment of 18232 disorders canbe brought about by techniques that serve to inhibit the expression oractivity of target gene products. For example, compounds, e.g., an agentidentified using assays described above, that exhibits negativemodulatory activities, can be used in accordance with the invention toprevent and/or ameliorate symptoms of 18232 disorders. Such moleculescan include, but are not limited to peptides, phosphopeptides, smallorganic or inorganic molecules, or antibodies (including, for example,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or singlechain antibodies, and FAb, F(ab′)₂ and FAb expression library fragments,scFV molecules, and epitope-binding fragments thereof).

[0395] Further, antisense and ribozyme molecules that inhibit expressionof the target gene can also be used in accordance with the invention toreduce the level of target gene expression, thus effectively reducingthe level of target gene activity. Still further, triple helix moleculescan be utilized in reducing the level of target gene activity.Antisense, ribozyme and triple helix molecules are discussed above.

[0396] It is possible that the use of antisense, ribozyme, and/or triplehelix molecules to reduce or inhibit mutant gene expression can alsoreduce or inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene alleles,such that the concentration of normal target gene product present can belower than is necessary for a normal phenotype. In such cases, nucleicacid molecules that encode and express target gene polypeptidesexhibiting normal target gene activity can be introduced into cells viagene therapy method. Alternatively, in instances in which the targetgene encodes an extracellular protein, it can be preferable toco-administer normal target gene protein into the cell or tissue inorder to maintain the requisite level of cellular or tissue target geneactivity.

[0397] Another method by which nucleic acid molecules may be utilized intreating or preventing a disease characterized by 18232 expression isthrough the use of aptamer molecules specific for 18232 protein.Aptamers are nucleic acid molecules having a tertiary structure thatpermits them to specifically bind to protein ligands (see, e.g.,Osborne, et al. 1997 Curr. Opin. Chem Biol. 1(1): 5-9; and Patel, D. J.1997 Curr Opin Chem Biol Jun;1(1):32-46). Since nucleic acid moleculesmay in many cases, be more conveniently introduced into target cellsthan therapeutic protein molecules, aptamers offer a method by which18232 protein activity may be specifically decreased without theintroduction of drugs or other molecules which may have pluripotenteffects.

[0398] Antibodies can be generated that are both specific for targetgene products and that reduce target gene product activity. Suchantibodies may, therefore, by administered in instances whereby negativemodulatory techniques are appropriate for the treatment of 18232disorders. For a description of antibodies, see the Antibody sectionabove.

[0399] In circumstances wherein injection of an animal or a humansubject with a 18232 protein or epitope for stimulating antibodyproduction is harmful to the subject, it is possible to generate animmune response against 18232 through the use of anti-idiotypicantibodies (see, for example, Herlyn, D. 1999 Ann Med 31(1):66-78; andBhattacharya-Chatterjee, M., and Foon, K. A. 1998 Cancer Treat Res94:51-68). If an anti-idiotypic antibody is introduced into a mammal orhuman subject, it should stimulate the production of anti-anti-idiotypicantibodies, which should be specific to the 18232 protein. Vaccinesdirected to a disease characterized by 18232 expression may also begenerated in this fashion.

[0400] In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0401] The identified compounds that inhibit target gene expression,synthesis and/or activity can be administered to a patient attherapeutically effective doses to prevent, treat or ameliorate 18232disorders. A therapeutically effective dose refers to that amount of thecompound sufficient to result in amelioration of symptoms of thedisorders.

[0402] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ and the ED₅₀ asdescribed above in the Pharmaceutical Composition section.

[0403] Another example of determination of effective dose for anindividual is the ability to directly assay levels of “free” and “bound”compound in the serum of the test subject. Such assays may utilizeantibody mimics and/or “biosensors” that have been created throughmolecular imprinting techniques. A compound that is able to modulate18232 activity is used as a template or “imprinting molecule”, tospatially organize polymerizable monomers prior to their polymerizationwith catalytic reagents. The subsequent removal of the imprintedmolecule leaves a polymer matrix that contains a repeated “negativeimage” of the compound and is able to selectively rebind the moleculeunder biological assay conditions. A detailed review of this techniquecan be seen in Ansell, R. J. et al (1996) Current Opinion inBiotechnology 7:89-94 and in Shea, K. J. (1994) Trends in PolymerScience 2:166-173. Such “imprinted” affinity matrixes are amenable toligand-binding assays, whereby the immobilized monoclonal antibodycomponent is replaced by an appropriately imprinted matrix. An exampleof the use of such matrixes in this way can be seen in Vlatakis, G. etal (1993) Nature 361:645-647. Through the use of isotope-labeling, the“free” concentration of compound which modulates the expression oractivity of 18232 can be readily monitored and used in calculations ofIC₅₀.

[0404] Such “imprinted” affinity matrixes can also be designed toinclude fluorescent groups whose photon-emitting properties measurablychange upon local and selective binding of target compound. Thesechanges can be readily assayed in real time using appropriate fiberopticdevices, in turn allowing the dose in a test subject to be quicklyoptimized based on its individual IC₅₀. A rudimentary example of such a“biosensor” is discussed in Kriz, D. et al (1995) Analytical Chemistry67:2142-2144.

[0405] Another aspect of the invention pertains to methods of modulating18232 expression or activity for therapeutic purposes. Accordingly, inan exemplary embodiment, the modulatory method of the invention involvescontacting a cell with 18232 or agent that modulates one or more of theactivities of 18232 protein activity associated with the cell. An agentthat modulates 18232 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 18232 protein (e.g., a 18232 substrate orreceptor), a 18232 antibody, a 18232 agonist or antagonist, apeptidomimetic of a 18232 agonist or antagonist, or other smallmolecule.

[0406] In one embodiment, the agent stimulates one or more 18232activities. Examples of such stimulatory agents include active 18232protein and a nucleic acid molecule encoding 18232. In anotherembodiment, the agent inhibits one or more 18232 activities. Examples ofsuch inhibitory agents include antisense 18232 nucleic acid molecules,anti-18232 antibodies, and 18232 inhibitors. These modulatory methodscan be performed in vitro (e.g., by culturing the cell with the agent)or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 18232 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) 18232 expression or activity. In anotherembodiment, the method involves administering a 18232 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted 18232 expression or activity.

[0407] Stimulation of 18232 activity is desirable in situations in which18232 is abnormally downregulated and/or in which increased 18232activity is likely to have a beneficial effect. For example, stimulationof 18232 activity is desirable in situations in which a 18232 isdownregulated and/or in which increased 18232 activity is likely to havea beneficial effect. Likewise, inhibition of 18232 activity is desirablein situations in which 18232 is abnormally upregulated and/or in whichdecreased 18232 activity is likely to have a beneficial effect.

[0408] Pharmacogenomics

[0409] The 18232 molecules of the present invention, as well as agents,or modulators which have a stimulatory or inhibitory effect on 18232activity (e.g., 18232 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 18232-associated disordersassociated with aberrant or unwanted 18232 activity (e.g.,hyperproliferative disorders, e.g., cancer). In conjunction with suchtreatment, pharmacogenomics may be considered. “Pharmacogenomics”, asused herein, refers to the application of genomics technologies such asgene sequencing, statistical genetics, and gene expression analysis todrugs in clinical development and on the market. More specifically, theterm refers the study of how a patient's genes determine his or herresponse to a drug (e.g., a patient's “drug response phenotype”, or“drug response genotype”.) Thus, another aspect of the inventionprovides methods for tailoring an individual's prophylactic ortherapeutic treatment with either the 18232 molecules of the presentinvention or 18232 modulators according to that individual's drugresponse genotype.

[0410] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum, M. etal. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder,M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms.

[0411] Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer a44576 molecule or 44576 modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a 44576 molecule or 44576modulator.

[0412] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

[0413] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drug's target is known (e.g., a18232 protein of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

[0414] Alternatively, a method termed “gene expression profiling”, canbe utilized to identify genes that predict drug response. For example,the gene expression of an animal dosed with a drug (e.g., a 18232molecule or 18232 modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

[0415] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment of anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with a18232 molecule or 18232 modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

[0416] The present invention further provides methods for identifyingnew agents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 18232 genes of the present invention, wherein theseproducts may be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 18232genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., cancer cells,will become sensitive to treatment with an agent that the unmodifiedtarget cells were resistant to.

[0417] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a 18232 protein can be applied in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase 18232 gene expression,protein levels, or upregulate 18232 activity, can be monitored inclinical trials of subjects exhibiting decreased 18232 gene expression,protein levels, or downregulated 18232 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decrease18232 gene expression, protein levels, or downregulate 18232 activity,can be monitored in clinical trials of subjects exhibiting increased18232 gene expression, protein levels, or upregulated 18232 activity. Insuch clinical trials, the expression or activity of a 18232 gene, andpreferably, other genes that have been implicated in, for example, a18232-associated disorder can be used as a “read out” or markers of thephenotype of a particular cell.

[0418] Other Embodiments

[0419] In another aspect, the invention features, a method of analyzinga plurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a 18232,preferably purified, nucleic acid, preferably purified, polypeptide,preferably purified, or antibody, and thereby evaluating the pluralityof capture probes. Binding, e.g., in the case of a nucleic acid,hybridization with a capture probe at an address of the plurality, isdetected, e.g., by signal generated from a label attached to the 18232nucleic acid, polypeptide, or antibody.

[0420] The capture probes can be a set of nucleic acids from a selectedsample, e.g., a sample of nucleic acids derived from a control ornon-stimulated tissue or cell.

[0421] The method can include contacting the 18232 nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

[0422] The plurality of capture probes can be a plurality of nucleicacid probes, each of which specifically hybridizes, with an allele of18232. Such methods can be used to diagnose a subject, e.g., to evaluaterisk for a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder. 18232 is associated with regulation of cell growth andproliferation, thus it is useful for evaluating, e.g., proliferationdisorders.

[0423] The method can be used to detect SNPs, as described above.

[0424] In another aspect, the invention features, a method of analyzinga plurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express 18232 or from a cell or subject in whicha 18232 mediated response has been elicited, e.g., by contact of thecell with 18232 nucleic acid or protein, or administration to the cellor subject 18232 nucleic acid or protein; contacting the array with oneor more inquiry probes, wherein an inquiry probe can be a nucleic acid,polypeptide, or antibody (which is preferably other than 18232 nucleicacid, polypeptide, or antibody); providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g.,wherein the capture probes are from a cell or subject which does notexpress 18232 (or does not express as highly as in the case of the 18232positive plurality of capture probes) or from a cell or subject which inwhich a 18232 mediated response has not been elicited (or has beenelicited to a lesser extent than in the first sample); contacting thearray with one or more inquiry probes (which is preferably other than a18232 nucleic acid, polypeptide, or antibody), and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by signal generated from a label attached to thenucleic acid, polypeptide, or antibody.

[0425] In another aspect, the invention features a method of analyzing18232, e.g., analyzing structure, function, or relatedness to othernucleic acid or amino acid sequences. The method includes: providing a18232 nucleic acid or amino acid; comparing the 18232 sequence with oneor more preferably a plurality of sequences from a collection ofsequences, e.g., a nucleic acid or protein sequence database; to therebyanalyze 18232. Preferred databases include GenBank. The method caninclude evaluating the sequence identity between a 18232 sequence and adatabase sequence. The method can be performed by accessing the databaseat a second site, e.g., over the internet.

[0426] In another aspect, the invention features, a set ofoligonucleotides, useful, e.g., for identifying SNP's, or identifyingspecific alleles of 18232. The set includes a plurality ofoligonucleotides, each of which has a different nucleotide at aninterrogation position, e.g., an SNP or the site of a mutation. In apreferred embodiment, the oligonucleotides of the plurality areidentical in sequence with one another (except for differences inlength). The oligonucleotides can be provided with differential labels,such that an oligonucleotide that hybridizes to one allele provides asignal that is distinguishable from an oligonucleotide that hybridizesto a second allele.

[0427] This invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human 18232cDNA

[0428] The human 18232 sequence (FIG. 1; SEQ ID NO:1), which isapproximately 1390 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 531nucleotides (nucleotides 329 to 859 of SEQ ID NO:1; SEQ ID NO:3). Thecoding sequence encodes a 176 amino acid protein (SEQ ID NO:2).

Example 2 Tissue Distribution of 18232 mRNA

[0429] Endogenous human 18232 gene expression was determined using thePerkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMantechnology. Briefly, TaqMan technology relies on standard RT-PCR withthe addition of a third gene-specific oligonucleotide (referred to as aprobe) which has a fluorescent dye coupled to its 5′ end (typically6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When thefluorescently tagged oligonucleotide is intact, the fluorescent signalfrom the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolyticactivity of Taq polymerase digests the labeled primer, producing a freenucleotide labeled with 6-FAM, which is now detected as a fluorescentsignal. The PCR cycle where fluorescence is first released and detectedis directly proportional to the starting amount of the gene of interestin the test sample, thus providing a way of quantitating the initialtemplate concentration. Samples can be internally controlled by theaddition of a second set of primers/probe specific for a housekeepinggene such as GAPDH which has been labeled with a different fluorophoreon the 5′ end (typically VIC).

[0430] To determine the level of 18232 in various human tissues aprimer/probe set was designed using Primer Express (Perkin-Elmer)software and primary cDNA sequence information. Total RNA was preparedfrom a series of human tissues using an RNeasy kit from Qiagen. Firststrand cDNA was prepared from 1 μg total RNA using an oligo-dT primerand Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained fromapproximately 50 ng total RNA was used per TaqMan reaction.

[0431] 18232 mRNA levels were analyzed in a variety of samples ofisolated and/or treated hematopoietic cells, in particular,hematopoietic progenitor CD34 cells. Expression was then furtherrestricted primarily to the erythroid lineage and increases as bonemarrow/blood cell differentiation proceeds.

[0432] High levels of 18232 mRNA expression, i.e., greater than 200relative units, was observed in skeletal muscle, fetal liver, and bonemarrow with low GPA levels (FIG. 4), and in late stage erythroid cells(Ery d10, d12) (FIG. 5). Moderate to high level mRNA expression, i.e.,greater than 100 relative units, was observed in lung tissue, indifferentiating erythroid cells, and in CD36+ erythroid cells (d14)(FIG. 5).

[0433]FIG. 5 shows relative 18232 mRNA expression on mRNA derived fromhuman hematological samples, e.g., bone marrow (BM), erythroid cells(ery), megakaryocytes (meg), neutrophils, and a negative referencesample (NTC). In some samples, mRNA expression was detected at theindicated times in culture (e.g., 24 hrs., 48 hrs., days in culture).High levels of 18232 mRNA expression, i.e., greater than 200 relativeunits, were observed in one sample of CD 15+, Cd11B− bone marrow; inerythroid cells, especially day 14 erythroid cells, glycophorin A orCD36+ erythroid cells; and in day 12 and day 14 megakaryocytes.

[0434]FIG. 6 shows relative 18232 mRNA expression on mRNA derived fromhuman tissue samples, both normal, and tumor. The samples are derivedfrom epithelial cells, colon, kidney, liver, fetal liver, lung, spleen,tonsil, lymph node, thymus, endothelial cells, skeletal muscle,fibroblast, and skin. The highest relative expression among thesesamples was found in epithelial cells, kidney, fetal liver, lung, andskeletal muscle.

[0435]FIG. 7 shows relative 18232 mRNA expression on mRNA derived fromadipose tissue, osteoblasts, aortic smooth muscle cells, and humanumbilical vein endothelial cells (HUVEC). FIG. 8 shows relative 18232mRNA expression in a variety of blood cells, e.g., CD8 positive cells,bone marrow cells, and cell culture lines (e.g., HepG2B, MA101, HL60,K562, and Hep3B).

[0436] Northern blot hybridizations with various RNA samples can beperformed under standard conditions and washed under stringentconditions, i.e., 0.2xSSC at 65° C. A DNA probe corresponding to all ora portion of the 18232 cDNA (SEQ ID NO:1) can be used. The DNA can beradioactively labeled with ³²P-dCTP using the Prime-It Kit (Stratagene,La Jolla, Calif.) according to the instructions of the supplier. Filterscontaining mRNA from mouse hematopoietic and endocrine tissues, andcancer cell lines (Clontech, Palo Alto, Calif.) can be probed inExpressHyb hybridization solution (Clontech) and washed at highstringency according to manufacturer's recommendations.

Example 3 Recombinant Expression of 18232 in Bacterial Cells

[0437] In this example, 18232 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 18232 isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-18232 fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 4 Expression of Recombinant 18232 Protein in COS Cells

[0438] To express the 18232 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 18232 protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

[0439] To construct the plasmid, the 18232 DNA sequence is amplified byPCR using two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 18232coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 18232 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 18232 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

[0440] COS cells are subsequently transfected with the 18232-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the 18232 polypeptide is detected byradiolabeling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

[0441] Alternatively, DNA containing the 18232 coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the 18232polypeptide is detected by radiolabelling and immunoprecipitation usinga 18232 specific monoclonal antibody.

[0442] Equivalents

[0443] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 5 1 1390 DNA Homo sapiens CDS (329)..(859) misc_feature (1)..(1390) n= a, t, g, or c 1 ccacgcgtcc ggcccagtgc ccaggccgcg ggggcgggga ggacggcgcccggggacaga 60 gaacatggga cgcagagcgg tccaaggccc cggcgccctg gtgaggcccaaacctcccgc 120 catgccccgg ccccaacgag acccaagccc cctgtcccgg cccagcgcccgcgggggacc 180 caagccccag cctggtccac ctcggaggcc tctaggaccc gggggcgcccggcggcccgc 240 ccggctccca caaatagact cctgggcggg cgcctgagcc cccaaaatagatcctcaggg 300 cccaaaagca gactcttcgg cgggcgcc atg gga ccg gca gaa gctggg cgc 352 Met Gly Pro Ala Glu Ala Gly Arg 1 5 cgc ggg gcc gcc tcg cccgta cct cca ccg ttg gtg cgc gtc gcg ccc 400 Arg Gly Ala Ala Ser Pro ValPro Pro Pro Leu Val Arg Val Ala Pro 10 15 20 tca ctc ttc ctc ggg agc gcgcga gcc gcg ggc gcg gag gag cag ctg 448 Ser Leu Phe Leu Gly Ser Ala ArgAla Ala Gly Ala Glu Glu Gln Leu 25 30 35 40 gcg cgc gcg gga gtc acg ctgtgc gtc aac gtc tcc cgc cag cag ccc 496 Ala Arg Ala Gly Val Thr Leu CysVal Asn Val Ser Arg Gln Gln Pro 45 50 55 ggc ccg cgc gcg ccc ggc gtg gcagag ctg cgc gtg ccc gtg ttc gac 544 Gly Pro Arg Ala Pro Gly Val Ala GluLeu Arg Val Pro Val Phe Asp 60 65 70 gac ccg gct gag gac ctg ctg gcg cacctg gag ccc acg tgc gcc gcc 592 Asp Pro Ala Glu Asp Leu Leu Ala His LeuGlu Pro Thr Cys Ala Ala 75 80 85 atg gag gcc gcg gtg cgc gcc ggc ggc gcctgc cta gtc tac tgc aag 640 Met Glu Ala Ala Val Arg Ala Gly Gly Ala CysLeu Val Tyr Cys Lys 90 95 100 aac ggc cgc agc cgc tcg gcc gcc gtc tgcacc gcg tac ctc atg cgg 688 Asn Gly Arg Ser Arg Ser Ala Ala Val Cys ThrAla Tyr Leu Met Arg 105 110 115 120 cac cgc ggc ctc agc ctg gcg aag gccttc cag atg gtg aag agc gct 736 His Arg Gly Leu Ser Leu Ala Lys Ala PheGln Met Val Lys Ser Ala 125 130 135 cgc ccg gta gca gaa ccg aac ccg ggcttc tgg tct cag ctc cag aag 784 Arg Pro Val Ala Glu Pro Asn Pro Gly PheTrp Ser Gln Leu Gln Lys 140 145 150 tat gag gag gcc ctc cag gcc cag tcctgc ctg cag gga gag ccc cca 832 Tyr Glu Glu Ala Leu Gln Ala Gln Ser CysLeu Gln Gly Glu Pro Pro 155 160 165 gcc tta ggg ttg ggc cct gag gct tgaagcttgaagg cytgctgcct 879 Ala Leu Gly Leu Gly Pro Glu Ala 170 175ggaggaagga tgtccctgca ctgatacaga agctgtttct ggcaaagcct gccgtgtctt 939acatttgtct ctctatccgg attagatgtt gctatatgaa cacatcggga ctgtgtctgc 999aggaaggagc tccccattcg aggccttcac agtgtcaccc acattcacct ctttccactt 1059aaacgtgtcc catgaatctt gtcataacag ttttgtgttc cttaactatt ttgtctgcca 1119tgtcatttat gatgtatata acctctttaa tgcctgaaat cataagaata atcatcaaag 1179gcaagagggt tgtatatttt cccgttggag acacatctgg aatttgctgc aataaaataa 1239taataagaaa gcnnaaaaaa aaaaaaaaaa aaaarmagna kkcaaaaakc gaggkagawa 1299tragcacacc gcttgtcttg ggctggacat ataattgctg gctggtgggt tgcaagaaat 1359ttctcttcaa gcatcatcac ccacttttgc t 1390 2 176 PRT Homo sapiensmisc_feature (1)..(1390) n = a, t, g, or c 2 Met Gly Pro Ala Glu Ala GlyArg Arg Gly Ala Ala Ser Pro Val Pro 1 5 10 15 Pro Pro Leu Val Arg ValAla Pro Ser Leu Phe Leu Gly Ser Ala Arg 20 25 30 Ala Ala Gly Ala Glu GluGln Leu Ala Arg Ala Gly Val Thr Leu Cys 35 40 45 Val Asn Val Ser Arg GlnGln Pro Gly Pro Arg Ala Pro Gly Val Ala 50 55 60 Glu Leu Arg Val Pro ValPhe Asp Asp Pro Ala Glu Asp Leu Leu Ala 65 70 75 80 His Leu Glu Pro ThrCys Ala Ala Met Glu Ala Ala Val Arg Ala Gly 85 90 95 Gly Ala Cys Leu ValTyr Cys Lys Asn Gly Arg Ser Arg Ser Ala Ala 100 105 110 Val Cys Thr AlaTyr Leu Met Arg His Arg Gly Leu Ser Leu Ala Lys 115 120 125 Ala Phe GlnMet Val Lys Ser Ala Arg Pro Val Ala Glu Pro Asn Pro 130 135 140 Gly PheTrp Ser Gln Leu Gln Lys Tyr Glu Glu Ala Leu Gln Ala Gln 145 150 155 160Ser Cys Leu Gln Gly Glu Pro Pro Ala Leu Gly Leu Gly Pro Glu Ala 165 170175 3 531 DNA Homo sapiens 3 atgggaccgg cagaagctgg gcgccgcggg gccgcctcgcccgtacctcc accgttggtg 60 cgcgtcgcgc cctcactctt cctcgggagc gcgcgagccgcgggcgcgga ggagcagctg 120 gcgcgcgcgg gagtcacgct gtgcgtcaac gtctcccgccagcagcccgg cccgcgcgcg 180 cccggcgtgg cagagctgcg cgtgcccgtg ttcgacgacccggctgagga cctgctggcg 240 cacctggagc ccacgtgcgc cgccatggag gccgcggtgcgcgccggcgg cgcctgccta 300 gtctactgca agaacggccg cagccgctcg gccgccgtctgcaccgcgta cctcatgcgg 360 caccgcggcc tcagcctggc gaaggccttc cagatggtgaagagcgctcg cccggtagca 420 gaaccgaacc cgggcttctg gtctcagctc cagaagtatgaggaggccct ccaggcccag 480 tcctgcctgc agggagagcc cccagcctta gggttgggccctgaggcttg a 531 4 173 PRT Artificial Sequence VARIANT (1)..(173)consensus sequence 4 Gly Pro Ser Glu Ile Leu Pro His Leu Tyr Leu Gly SerTyr Ser Thr 1 5 10 15 Ala Ser Glu Ala Asn Leu Ala Leu Leu Lys Lys LeuGly Ile Thr His 20 25 30 Val Ile Asn Val Thr Glu Glu Val Pro Asn Pro PheGlu Leu Asp Lys 35 40 45 Lys Asn Asp Arg His Tyr Thr Asn Ala Tyr Ile SerLys Asn Ser Gly 50 55 60 Phe Thr Tyr Leu Gln Ile Pro Asn Val Asp Asp HisIle Tyr Tyr His 65 70 75 80 Ile Ala Trp Asn His Glu Thr Lys Ile Ser LysTyr Phe Asp Glu Ala 85 90 95 Val Asp Phe Ile Asp Asp Ala Arg Gln Lys GlyGly Lys Val Leu Val 100 105 110 His Cys Gln Ala Gly Ile Ser Arg Ser AlaThr Leu Ile Ile Ala Tyr 115 120 125 Leu Met Lys Thr Arg Asn Leu Ser LeuAsn Glu Ala Tyr Asp Phe Val 130 135 140 Tyr Val Tyr His Ile Lys Glu ArgArg Cys Pro Ile Ile Ser Pro Asn 145 150 155 160 Phe Gly Phe Leu Arg GlnLeu Ile Glu Tyr Glu Arg Lys 165 170 5 172 PRT Artificial SequenceVARIANT (1)..(172) consensus sequence 5 Gly Pro Ser Glu Ile Leu Pro HisLeu Tyr Leu Gly Ser Tyr Ser Asp 1 5 10 15 Ala Ser Glu Ala Asn Leu AlaLeu Leu Lys Lys Leu Gly Ile Thr His 20 25 30 Val Ile Asn Val Thr Glu GluVal Pro Asn Asn Phe Glu Leu Lys Lys 35 40 45 Lys Asn Asp Arg Tyr Tyr ThrAsn Glu Tyr Ile Ser Lys Gly Ser Gly 50 55 60 Phe Thr Tyr Leu Gln Ile ProAsn Val Asp Asp Ile Tyr Tyr His Ile 65 70 75 80 Ala Trp Asn Thr Glu ThrLys Ile Ser Lys Tyr Leu Glu Glu Ala Val 85 90 95 Glu Phe Ile Glu Asp AlaGlu Lys Lys Gly Gly Lys Val Leu Val His 100 105 110 Cys Gln Ala Gly ValSer Arg Ser Ala Thr Leu Val Ile Ala Tyr Leu 115 120 125 Met Lys Thr ArgAsn Leu Ser Leu Arg Asp Ala Tyr Asp Phe Val Tyr 130 135 140 Val Tyr HisIle Lys Glu Arg Arg Cys Pro Ile Ile Ser Pro Asn Phe 145 150 155 160 GlyPhe Leu Arg Gln Leu Ile Glu Tyr Glu Arg Lys 165 170

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: a) a nucleic acid molecule comprising anucleotide sequence which is at least 60% identical to the nucleotidesequence of SEQ ID NO:1, 3, or the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ______; b) a nucleic acid moleculecomprising a fragment of at least 381 nucleotides of the nucleotidesequence of SEQ ID NO:1, 3, or the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ______; c) a nucleic acid moleculewhich encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:2, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number ______; d) a nucleicacid molecule which encodes a fragment of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with the ATCC as AccessionNumber ______, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:2, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession Number______; and e) a nucleic acid molecule which encodes a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Accession Number______, wherein the nucleic acid molecule hybridizes to a nucleic acidmolecule comprising SEQ ID NO:1, 3, or a complement thereof, understringent conditions.
 2. The isolated nucleic acid molecule of claim 1,which is selected from the group consisting of: a) a nucleic acidcomprising the nucleotide sequence of SEQ ID NO:1, 3, or the cDNA insertof the plasmid deposited with the ATCC as Accession Number ______; andb) a nucleic acid molecule which encodes a polypeptide comprising theamino acid sequence of SEQ ID NO:2, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with the ATCC as AccessionNumber ______.
 3. The nucleic acid molecule of claim 1 furthercomprising vector nucleic acid sequences.
 4. The nucleic acid moleculeof claim 1 further comprising nucleic acid sequences encoding aheterologous polypeptide.
 5. A host cell which contains the nucleic acidmolecule of claim
 1. 6. The host cell of claim 5 which is a mammalianhost cell.
 7. A non-human mammalian host cell containing the nucleicacid molecule of claim
 1. 8. An isolated polypeptide selected from thegroup consisting of: a) a polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence which is at least 60%identical to a nucleic acid comprising the nucleotide sequence of SEQ IDNO:1, 3, the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number ______, or acomplement thereof; b) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ______, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:1, 3, or a complement thereof understringent conditions; and c) a fragment of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, or the amino acid sequence encodedby the cDNA insert of the plasmid deposited with the ATCC as AccessionNumber ______, wherein the fragment comprises at least 15 contiguousamino acids of SEQ ID NO:2.
 9. The isolated polypeptide of claim 8comprising the amino acid sequence of SEQ ID NO:2.
 10. The polypeptideof claim 8 further comprising heterologous amino acid sequences.
 11. Anantibody which selectively binds to a polypeptide of claim
 8. 12. Amethod for producing a polypeptide selected from the group consistingof: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2,or the amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as Accession Number ______; b) a polypeptidecomprising a fragment of the amino acid sequence of SEQ ID NO:2, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ______, wherein the fragment comprisesat least 15 contiguous amino acids of SEQ ID NO:2, or the amino acidsequence encoded by the cDNA insert of the plasmid deposited with theATCC as Accession Number ______; and c) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:2, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number ______, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising SEQ ID NO:1, 3, or a complement thereofunder stringent conditions; comprising culturing the host cell of claim5 under conditions in which the nucleic acid molecule is expressed. 13.A method for detecting the presence of a polypeptide of claim 8 in asample, comprising: a) contacting the sample with a compound whichselectively binds to a polypeptide of claim 8; and b) determiningwhether the compound binds to the polypeptide in the sampl
 14. A kitcomprising a compound which selectively binds to a polypeptide of claim8 and instructions for use.
 15. A method for detecting the presence of anucleic acid molecule of claim 1 in a sample, comprising the steps of:a) contacting the sample with a nucleic acid probe or primer whichselectively hybridizes to the nucleic acid molecule; and b) determiningwhether the nucleic acid probe or primer binds to a nucleic acidmolecule in the sample.
 16. A kit comprising a compound whichselectively hybridizes to a nucleic acid molecule of claim 1 andinstructions for use.
 17. A method for identifying a compound whichbinds to a polypeptide of claim 8 comprising the steps of: a) contactinga polypeptide, or a cell expressing a polypeptide of claim 8 with a testcompound; and b) determining whether the polypeptide binds to the testcompound.
 18. The method of claim 17, wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor tyrosine phosphatase-mediated signal transduction.
 19. A method foridentifying a compound which modulates the activity of a polypeptide ofclaim 8, comprising: a) contacting a polypeptide of claim 8 with a testcompound; and b) determining the effect of the test compound on theactivity of the polypeptide to thereby identify a compound whichmodulates the activity of the polypeptide.
 20. A method for modulatinghematopoiesis, comprising contacting a hematopoietic cell with an agentthat modulates the activity or expression of a polypeptide of claim 8,thereby modulating the proliferation, differentiation or survival of thehematopoietic cell.
 21. The method of claim 20, wherein thehematopoietic cell is a CD34-expressing cell.
 22. A method of modulatinghematopoiesis, comprising contacting a hematopoietic cell with an agentthat modulates the activity or expression of a nucleic acid of claim 1,thereby modulating the proliferation, differentiation or survival of thehematopoietic cell.
 23. A method of treating or preventing anerythroid-associated disorder, in a subject, comprising administering tothe subject an effective amount of a compound that modulates theactivity or expression of a polypeptide of claim 8, such that theerythroid-associated disorder is ameliorated or prevented.
 24. A methodfor evaluating the efficacy of a treatment of an erythroid-associateddisorder, in a subject, comprising: treating a subject with a protocolunder evaluation; assessing the expression level of a nucleic acid ofclaim 1, or a polypeptide of claim 8, wherein a change in the expressionlevel of the nucleic acid or the polypeptide after the treatment,relative to the level before the treatment, is indicative of theefficacy of the treatment of the erythroid-associated disorder.
 25. Themethod of claim 24, further comprising treating the subject witherythropoietin prior to assessing expression levels.
 26. A method ofdiagnosing an erythroid-associated disorder, in a subject, comprising:evaluating the expression or activity of a nucleic acid of claim 1, or apolypeptide of claim 8, such that, a difference in the level of thenucleic acid or the polypeptide relative to a normal subject or a cohortof normal subjects is indicative of the erythroid-associated disorder.